Escherichia coli K-12 ampicillin-resistant mutants hyperproducing chromosomal β-lactamase arose spontaneously from strains carrying ampA1 ampC+. Such mutants were found even in a recA background. Two Ampr-100 strains were analyzed genetically. The Ampr-100 resistance level of both strains could be transduced by direct selection for ampicillin resistance. Several classes of ampicillin-resistant transductants were found that differed from one another in the β-lactamase activity and the ampicillin resistance mediated by an ampA1 ampC+-carrying strain. The data suggested that β-lactamase hyperproduction was due to repetitions of the chromosomal amp genes. The size of the repeated region was calculated from cotransduction estimates, using the formula of Wu (Genetics 54:405-410, 1966), and was found to be about 1 min in one strain and 1.5 min in the other. Second-step Ampr-400 mutants were isolated from an Ampr-100 strain. The resistance of these mutants was apparently also due to repetitions, each mediating a resistance to about 10 μg/ml. Mutants of wild-type strains that were moderately resistant to ampicillin also gave rise to intermediate-resistance classes, suggesting repetitions of the wild-type amp alleles. F′ factors hyperproducing chromosomal β-lactamase by gene repetitions were constructed. They mediated levels of ampicillin resistance comparable to that of naturally occurring resistance plasmids. The expression of β-lactamase hyperproduction was not affected by the presence of ampA and ampC alleles in trans and did not act in trans on the other alleles.