The influence of DNA repair on the specificity of ethyl methanesulfonate (EMS) mutagenesis in a plasmid-borne copy of the Saccharomyces cerevisiae SUP4-o gene was investigated. Isogenic yeast strains that are repair-proficient (RAD) or defective for nucleotide excision (rad1), postreplication (rad18) or recombinational repair (rad52) were treated with EMS. Compared to the RAD wild-type, the maximum SUP4-o mutation frequency was 2-fold greater in the rad1 background whereas it was approximately 50% less in the rad18 and rad52 strains. The majority (779/788) of SUP4-o mutations characterized by DNA sequencing were single base pair changes, primarily (> 91%) G.C-->A.T transitions in the RAD, rad1 and rad18 strains. In the rad52 background, only 57% of the substitutions were G.C-->A.T transitions with transversions at G.C pairs accounting for almost all of the remaining changes. Comparisons of the distributions of single base pair substitutions in SUP4-o revealed that there was no excision repair-dependent bias for G.C-->A.T events to occur at sites flanked by a 5' or 3' A.T pair as observed previously for EMS mutagenesis of the lacIgene in Escherichia coli (Burns et al., 1986). These transitions also did not occur more often at sites where the guanine was flanked by a 5' purine than by a 5' pyrimidine. However, they exhibited a small preference for sites having the guanine on the transcribed strand in the RAD and rad52, but not rad1 or rad18, strains.(ABSTRACT TRUNCATED AT 250 WORDS)