O6-Alkylguanine-DNA alkyltransferase (AGT) repairs O 6-alkylguanine residues at different rates depending on the identity of the alkyl group as well as the sequence context. To elucidate the mechanism(s) underlying the differences in rates, we examined the repair of five alkyl groups in three different sequence contexts. The kinact and Km values were determined by measuring the rates of repair of oligodeoxynucleotide duplexes containing the O6-alkylguanine residues with various concentrations of AGT in excess. The time course of the reactions all followed pseudo-first-order kinetics except for one of the O 6-ethylguanine substrates, which could be analyzed in a two-phase exponential equation. The differences in rates of repair between the different alkyl groups and the different sequence contexts are dependent on rates of alkyl transfer and not substrate recognition. The relative rates of reaction are in general benzyl > methyl > ethyl > 2-hydroxyethyl > 4-(3-pyridyl)-4-oxobutyl, but the absolute rates are dependent on sequence. The kinact values between benzyl and 4-(3-pyridyl)-4-oxobutyl range from 2300 to 350000 depending on sequence. The sequence-dependent variation in k inact varied the most for O6-[4-(3-pyridyl)-4-oxobutyl] guanine, which ranged from 0.022 to 0.000016 s-1. The results are consistent with a mechanism in which the O6-alkylguanine can bind to AGT in either a reactive or an unreactive orientation, the proportion of which depends on the sequence context.