Purpose: Determining the absorbed dose produced by photons, it is often assumed that it is equal to the radiation kerma. This assumption is valid only in the presence of an electronic equilibrium, which in turn is never ensured in practice. It leads to some uncertainty in determining the absorbed dose in the irradiated sample during radiobiological experiments. Therefore, it is necessary to estimate the uncertainty in determining the relative biological effectiveness of X-rays associated with uncertainty in the determination of the absorbed dose. Material and methods: The monochromatic X-ray photon emission is simulated through a standard 25 cm2 plastic flask containing 5 ml of the model culture medium (biological tissue with elemental composition C5H40O18N). The calculation of the absorbed dose in a culture medium is carried out in two ways: 1) the standard method, according to which the ratio of the absorbed dose in the medium and the ionization chamber is equal to the ratio of kerma in the medium and air; 2) determination of the absorbed dose in the medium and in the sensitive volume of the ionization chamber by computer simulation and calculating the ratio of these doses. For each primary photon energies, 108 histories are modeled, which makes it possible to achieve a statistical uncertainty not worse than 0.1 %. The energy step was 1 keV. The spectral distribution of X-ray energy is modeled separately for each set of anode materials, thickness and materials of the primary and secondary filters. The specification of the X-ray beams modeled in this work corresponds to the standards ISO 4037 and IEC 61267. Within the linear-quadratic model, the uncertainty of determining the RBEmax values is directly proportional to the uncertainty in the determination of the dose absorbed by the sample under study. Results: At energy of more than 60 keV, the ratios for water and biological tissue practically do not differ. At lower energies, up to about 20 keV, the ratio of the coefficients of air and water is slightly less than that of air and biological tissue. The maximum difference is ~ 1 % than usual and the equality of absorbed doses in the ionization chamber and sample is justified. At photon energy of 60 keV for the geometry in question, the uncertainty in determining the dose is about 50 %. For non-monochromatic radiation, the magnitude of the uncertainty is determined by the spectral composition of the radiation, since the curves vary greatly in the energy range 10–100 keV. It is shown that, depending on the spectral composition of X-ray radiation, uncertainty in the determination of the absorbed dose can reach 40–60 %. Such large uncertainty is due to the lack of electronic equilibrium in the radiation geometry used in practice. The spread of RBE values determined from the data of radiobiological experiments carried out by different authors can be determined both by differences in the experimental conditions and by uncertainty in the determination of the absorbed dose. Using Fricke dosimeters instead of ionization chambers in the same geometry allows you to reduce the uncertainty approximately 2 times, up to 10–30 %. Conclusion: The computer simulation of radiobiological experiments to determine the relative biological effectiveness of X-ray radiation is performed. The geometry of the experiments corresponds to the conditions for the use of standard bottles placed in the side holders. It is shown that the ratio of absorbed doses and kerma in the layers of biological tissue and air differ among themselves with an uncertainty up to 60 %. Depending on the quality of the beam, the true absorbed dose may differ from the one calculated on the assumption of kerma and dose equivalence by 50 %. Uncertainty in determining the RBE in these experiments is of the same order. The results are presented for X-ray beams with negligible fraction of photons with energies less than 10 keV. For beams of a different quality, the uncertainty in determination can significantly increase. For the correct evaluation of RBE, it is necessary to develop a uniform standard for carrying out radiobiological experiments. This standard should regulate both the geometry of the experiments and the conduct of dosimetric measurements.