The mutagenic potential of O4-methylthymine (m4T) and O4-ethylthymine (e4T) was determined by a primer extension assay on a 25mer oligonucleotide containing a single sitespecifically incorporated modified thymine. The e4T-containing oligonucleotide was prepared by using a new synthetic procedure suitable for large alkyl groups on thymine. The second-order rate constants, Kappm and Vrelmax, permitted calculation of the frequency of formation and extension of modified base pairs compared to Watson — Crick pairing. With both m4T and e4T, the T.G type pairing was formed at least 10-fold more frequently than the non-mutagenic alkyl T.A pairing. However, there was a small but reproducible preference for m4T.G pairing. In both cases T ↑ C transitions would result. There was no evidence for formation of alkyl T.C or T.T. These data suggest that reported T — A transversions by ethylation are not likely to result from O4-alkylthymine. In contrast to insertion, extension beyond alkylthymine under kinetic conditions did not occur with alkyl T.A but only with the alkyl T-G termini. For this latter T-G type pairing, the larger ethyl group did not hinder extension compared to that of the methyl group, in the sequence studied. Under non-limiting conditions of dNTP concentration and time, complete replication could be demonstrated for both methyl- and ethyl-containing oligonucleotides. We conclude that the increase in size of the alkyl group from methyl to ethyl does not significantly affect the mutagenic potential and type of mutations of O4-alkylthymine in vitro.