The role of the proofreading (3' leads to 5' exonuclease) function of T4 DNA polymerase and the mismatch repair system of E. coli on N4-hydroxycytidine (oh4Cyd) induced mutagenesis was investigated. oh4Cyd-induced mutation is strongly suppressed when the proofreading activity increases as a result of the presence of tsCB87--antimutator polymerase or elevated temperature (43 degrees C vs 30 degrees C). Mutagenic activity of oh4Cyd, however, is little, if at all, affected by the presence of the tsLB56 mutator allele of T4 DNA polymerase with suppressed proofreading activity. This leads to the conclusion that oh4Cyd nucleotides are not frequently removed by proofreading activity of wild-type T4 DNA polymerase. The number of mutations induced by oh4Cyd increases 3- to 5-fold due to damage of the genes mutS, mutL, uvrE, but not mutR. Dam- cells are more sensitive to, and hypermutable by, oh4Cyd in comparison with dam+ cells. This is compatible with the notion that oh4C residues are recognised and excised by mismatch repair enzymes. The results indicate that neither the proofreading function of T4 DNA polymerase, nor the mismatch repair enzymes, are responsible for the high specificity of oh4Cyd which causes AT leads to GC transition.