A novel mutator locus in Escherichia coli was identified from a collection of random transposon insertion mutants. Several mutators in this collection were found to have an insertion in the dgt gene, encoding a previously characterized dGTP triphosphohydrolase. The mutator activity of the dgt mutants displays an unusual specificity. Among the six possible base pair substitutions in a lacZ reversion system, the G.C-->C.G transversion and A.T-->G.C transition are strongly enhanced (10- to 50-fold), while a modest effect (two- to threefold) is also observed for the G.C-->A.T transition. Interestingly, a two- to threefold reduction in mutant frequency (antimutator effect) is observed for the G.C-->T.A transversion. In the absence of DNA mismatch repair (mutL) some of these effects are reduced or abolished, while other effects remain unchanged. Analysis of these effects, combined with the DNA sequence contexts in which the reversions take place, suggests that alterations of the dGTP pools as well as alterations in the level of some modified dNTP derivatives could affect the fidelity of in vivo DNA replication and, hence, account for the overall mutator effects.