Of four enzymatically active forms of Alcaligenes faecalis penicillin amidase (EC 3.5.1.11) observed in sonicated cells, two (PA(5.5) and PA(5.3); subscript denotes pI) could be isolated and purified in two steps from the cells destroyed by osmotic shock. Active enzyme was only found in the periplasm. PA(5.5) converts further to PA(5.3) which differs in the molecular mass of the A-chain. The origin of these differences is a conversion of the N-terminal Gln to pyrolidenocarboxilic acid and a loss of three amino acids from the C-terminus of the A-chain. PA(5.3) had higher specific activity (10-30%) for the hydrolysis of benzylpenicillin and 6-nitro-3-phenylacetamidobenzoic acid than PA(5.5).