Assuming that the efficiency of the incorporation of 5-methyl-2′-deoxyisocytosine-5′ triphosphate (dMiCTP) and dTTP opposite isoguanine (iG) is a measure of the proportion of the keto and enol tautomers of iG in the Thermus aquaticus DNA polymerase active centre, we studied the influence of temperature and iG-neighbouring bases in the template on base-pairing of iG. On the basis of experiments with four sequences (3′-TXT-5′, 3′-GXG-5′, 3′-CXC-5′, 3′-CXT-5′, where X = iG) at 37, 50, 65 and 80°C, we found that 3′-neighbours decrease the fraction of the keto tautomer in the order C>GT, whereas temperature apparently does not influence the tautomeric equilibrium of iG. The variability of the ratio of incorporation of dMiCTP versus dTTP (5—20) primarily reflects the variability of Km values, since Vmax values are roughly similar, which indicates that the iG·MiC and iG·T pairs fit the polymerase active centre equally well. The altering of the base-pairing of iG by sequence context is discussed in relation to tautomerism and miscoding of this oxidized adenine derivative. A key derivative for preparation oligodeoxynucleotides, O2-diphenylcarbamoyl-N6-[(dimethylamino)ethylidene]-2′-deoxyisoguanosine, is extremely labile (t1/2 = 3.5min) in 3% trichloroacetic acid/dichloromethane, i.e. under the conditions of automated DNA synthesis, which results in low yield and length heterogeneity of templates.