Glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas sp. GK16 produces glutaryl-7-aminocephalosporanic acid, a key intermediate for the synthesis of cephem antibiotics. Sequence alignment suggests that the enzyme may belong to the N-terminal nucleophile hydrolase superfamily including penicillin G acylase. The enzyme is an (alpha beta)(2) heterotetramer of two nonidentical subunits. These subunits are derived from a nascent precursor polypeptide that is cleaved proteolytically through a two-step autocatalytic process upon folding. The enzyme has been crystallized using the vapor diffusion method. A bipyramidal crystal form was obtained from a solution containing polyethylene glycol (MW 3350) and calcium chloride. Complete diffraction data sets have been collected up to 2.8 Angstrom resolution. The crystal is tetragonal with the space group P4(1)2(1)2 or P4(3)2(1)2 and the unit cell parameters are a = 5 = 73.5 Angstrom, c 380.3 Angstrom. Considerations of the possible values of V-m account for the presence of a tetramer in the asymmetric unit.