Abstract The triplet state of acridine orange dissolved in methanol/water matrix was investigated by ESR. In absence of oxygen a strong temperature dependence of the spectra was observed. At low temperature (100 °K) the zero-field splitting parameters calculated from the triplet spectrum are: X/hc = 0.0050 cm-1, Y/hc= 0.0342 cm-1, Z/hc=0.0387 cm-1 , at higher temperature (140 °K) : X*(hc=0.0056 cm-1, Y*/hc=0.0206 cm-1, Z*/hc = 0.0262 cm-1 . It was assumed that the low temperature spectrum is caused by isolated molecules in the triplet state while the high temperature spectrum must be attributed to the triplet exciton state of the acridine orange dimer. From the theory of the ESR triplet exciton spectra it can be shown that in the dimer state of acridine orange the molecular planes form an angle of 50° or 130°. However, it cannot be excluded that the dimer configuration differs in the ground or excited singlet state from the triplet state.