A simple biological assay to detect beta-lactamase activity exhibited by selected cultures of Escherichia coli was used to test enzyme production in cells incubated aerobically and anaerobically. Anaerobic incubation resulted in increased size of zones of drug inactivation by some beta-lactamase-producing strains. The beta-lactamase activity of cell lysates was determined iodometrically for aerobically and anaerobically grown cells. The specific beta-lactamase activity for anaerobically grown cells was three to five times greater than for aerobically grown cells. Beta-lactamase production was determined to be constitutive in all strains and to be plasmid mediated, as demonstrated by transfer to E. coli K-12 by conjugation.