Penicillin acylase is currently one of the most relevant industrial enzymes. The intracellular enzyme from Escherichia coli is used mostly, while a few processes use the extracellular enzyme from Bacillus megaterium. The production of the former has been thoroughly studied, but information on penicillin acylase production from B. megaterium is scarce. Results are presented for the production of penicillin acylase from B. megaterium in complex and defined media. The best conditions for induction by phenylacetic acid were determined and excretion of the enzyme was found to be strongly dependent on medium composition, a high level of inorganic phosphate being favourable for excretion. The enzyme produced was recovered, partially purified by membrane separation and characterized. The stability and kinetic behaviour were good, providing encouragement to pursue immobilization studies to develop an industrial quality catalyst.