Escherichia coli K-12 strain EY5, deficient in oxyR, was constructed to assess the role of oxyR and oxyR-regulated regulon in spontaneous mutagenesis. Mutagenesis was monitored by selecting two forward mutations of colicin B-sensitive to resistance and valine-sensitive to resistance, one base substitution mutation of rifampicin-sensitive to resistance and one reversion of argE3 his-4 to Arg(+) His(+). Deficiency of oxyR did not lead to the enhancement of spontaneous mutation frequencies of the four markers tested. By DNA sequence analysis, we determined 49 colicin B-resistant mutants derived from EY5 and found that 37% were base substitutions, 29% IS element insertions, 20% deletions, and 14% single base frameshifts. Among the base substitutions, G:C-->T:A transversions predominated followed by G:C-->A:T transitions and A:T-->T:A transversions. These spectra were essentially the same as those from oxyR(+) strains. The results indicate that oxyR and oxyR-regulated genes do not play a significant role in the defense against spontaneous mutagenesis.