In Enterobacter cloacae, mutations in favor of overproduction of beta-lactamase--leading to resistance to third-generation cephalosporins--occur at frequencies of 10(-4)-10(-7). Cloning experiments reveal that at least three genes are involved in the regulation of chromosomal beta-lactamase expression. The structural gene, ampC, is located adjacent to the regulatory gene, ampR, coding for a protein that can serve as an activator in the presence of an inducer. An example of an ampR mutant that is independent of an inducer has been studied. More important for the development of cefotaxime resistance in E. cloacae are mutations in the ampD gene and other proposed regulatory genes. Inactivation of the ampD gene leads to elevated beta-lactamase production. Thus, ampD negatively controls ampC expression. Evidence for the existence of a third regulatory gene, ampE, has been found.