The N-acetylglutamate deacetylase (EC 3.5.1.-) from Pseudomonas aeruginosa, strain PAO1, was purified 15,000-fold to electrophoretic homogeneity. The enzyme was distinct from acetylornithinase and formylglutamate hydrolase. Its molecular weight was estimated to be 90,000 by gel filtration and by sedimentation in sucrose gradients. Electrophoresis in sodium-dodecyl sulphate gels gave a single band corresponding to a molecular weight of 44,000. N-Acetylglutamate deacetylase was L-specific and showed no peptidase activity. Among 17 N-acetyl-L-amino acids tested as substrates, N-acetyl-L-glutamine, N-acetyl-L-methionine and N-acetylglycine were hydrolysed at 20% of the rate of N-acetyl-L-glutamate whereas other N-acetyl-L-amino acids were deacetylated at a rate of less than 10%. The catalytic activity depended on Co2+. The Km of the enzyme with respect to N-acetylglutamate was 1.43 mM. Preparation of spheroplasts with lysozyme in the presence of 0.2 M-MgCl2 led to the release of 80% of the enzyme activity from the cells, indicating the periplasmic localization of N-acetylglutamate deacetylase. Its localization in the periplasmic space explains the inability of P. aeruginosa argA mutants to grow on N-acetylglutamate, which is utilized by the wild-type as a carbon and nitrogen source.