Seven Escherichia coli K-12 mutants with a lowered chromosomal beta-lactamase activity were analyzed genetically. The beta-lactamase-negative mutants isolated from ampA1-carrying strains (resistant to 10 microgram of ampicillin per ml) all carried genetic lesions very close to the ampA1 mutation, which was still present. In an earlier report, two of the mutations mediating a beta-lactamase-negative phenotype (L. G. Burman, T. Park, E. B. Linström, and H. G. Boman, J. Bacteriol. 116:123-130, 1973) were shown to have occurred in the structural gene for beta-lactamase, designated ampC. It is suggested that all beta-lactamase-negative mutants studied here were altered in ampC. The relative order of ampC mutations was (ampC1, ampC8)-ampC9-(ampC12, ampC14)-ampC11, and the gene order was found to be ampC-1mpA-purA. The ampA1 allele was dominant over its wild-type allele but acted only cis and not trans, suggesting that ampA is the promoter or operator region for ampC. A gene dosage effect was found for strains homozygous for ampA+ ampC+ or ampA1 ampC+. Heterozygotes carrying the ampC8 allele on the chromosome showed an apparent derepression of the episomal ampC allele, suggesting a role for beta-lactamase in its own regulation.