Intact pZ189 DNA was allowed to replicate in monkey kidney vero cells that had been pretreated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The E. coli MBM7070 was transfected with replicated plasmid, and those with mutations in the supF gene were identified. The frequency of mutants that did not contain recognizable changes in the electrophoretic mobility of the plasmid DNA was scored. The frequency of such mutants was 12.2 x 10(-4) (43/35376) and 6.2 x 10(-4) (22/35712) in mutants derived from cells pretreated with 0.2 mumoles/l and 2 mumoles/l MNNG respectively; these values represent an increase of 5.8- and 2.9-fold over the spontaneous mutation frequency of 2.1 x 10(-4) (10/47741) (p < 0.01). Sequence analysis of the supF genes of these mutants showed that 89% (24/27) of base substitutions occurred at G.C base pairs; 59% of the base substitutions (16/27) were transversions, and 41% (11/27) were transitions. The types of base substitutions were predominantly G.C-->T.A and G.C-->A.T. 48% of base substitutions occurred at 6 sites of the supF gene; 4 of these sites consist of 5'-TTNN where N is G or C. Base substitutions never previously reported were found, namely, T-->C at 61, G-->T at 70, G-->T at 99, and G-->C at 103 were found; these have never been reported up to now. In addition, 2 of the 5 frameshifts occurred in the region 99-105 of the supF gene (GGTGGGG), suggesting that this region is a hot spot for nontargeted frameshifts. These results strongly suggest that nontargeted mutagenesis can occur in mammalian cells and shows that the spectrum of mutations induced differs from that of spontaneous and targeted mutations.