AbstractPenicillin amidase, an enzyme which hydrolyzes benzylpenicillin to 6‐aminopenicillanic acid and phenylacetic acid, is produced by Bacillus megaterium ATCC 14945 as an extracellular enzyme. We used this system as a model to examine the effects of nitrogen, sulfur, and phosphorous limitation on enzyme production in continuous culture. For these studies, we developed a minimal medium for B. megaterium which contained histidine as the sole nitrogen source. Batch experiments showed that this enzyme is produced as a growth‐associated metabolite. Enzyme production was shown to be a function of the growth‐limiting conditions and the concentration of the inducer, phenylacetic acid. Sulfur limitation in continuous culture yielded enzyme activities approximately three to five times those observed in nitrogen‐ and phosphorous‐limited chemostats. These results are discussed in terms of the environment's influence on enzyme production in continuous culture.