Benzylpenicillin acylase, which hydrolyzes benzylpenicillin to 6-aminopenicillanic acid, was found to be widely distributed among members of the Schizomycetes, particularly in gram-negative bacteria, and in the genus Nocardia. The hydrolysis of a series of biosynthetic and semisynthetic penicillins by freeze-dried cells of a strain of Nocardia and of Proteus was studied. Benzylpenicillin was the preferred substrate; all departures from the benzylpenicillin side-chain structure led to reduction of substrate activity (the greater the departure, the greater the reduction in activity). Penicillin amides and methyl esters were also hydrolyzed, as were suitable N-acyl derivatives of 7-aminocephalosporanic acid. Occurrence of an enzyme activity which hydrolyzes benzylpenicillinamide to benzylpenicillin was detected in certain strains of yeasts.