A mutant of Escherichia coli deficient in DNA polymerase II has been isolated from E. coli polA1 by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and assay of polymerase activity in extracts of survivors. The polA1 mutation was suppressed during mutagenesis by introduction of the suppressor, su7(+), into the parental strain. The mutant, HMS83 polA1 polB1, contains less than 0.5% of the normal levels of DNA polymerase II. The only polymerase activity detected in the mutant is DNA polymerase III. E. coli HMS83 grows normally at both 25 and 42 degrees , and supports the growth of bacteriophages T4, T7, lambda, varphiX174, 186, P2, and fd. The polB mutation does not affect sensitivity to ultraviolet irradiation or recombination frequencies.