Assuming that the efficiency of the incorporation of 5-methyl-2h-deoxyisocytosine-5h 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 iGneighbouring bases in the template on base-pairing of iG. On the basis of experiments with four sequences (3h-TXT-5h, 3h-GXG-5h, 3h-CXC-5h, 3h-CXT-5h, where X l iG) at 37, 50, 65 and 80 mC, we found that 3h-neighbours decrease the fraction of the keto tautomer in the order C G T, 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 K m values, since