The enzymatic profiles of 109 clinical isolates of Acinetobacter calcoaceticus subsp. anitratus and lwoffi were determined with conventional plate tests and the rapid API ZYM system (Analytab Products, Plainview, N.Y.). The majority of strains tested lacked DNase, hemolysin, protease, elastase and gelatinase. Strong enzymatic activities of butyrate esterase (C4), caprylate esterase (C8) and leucine arylamidase were detected in all isolates. No trypsin, chymotrypsin, alkaline phosphatase or glucosidase activities were present. This profile was characteristic of all isolates examined by the API ZYM system and could serve as a useful diagnostic feature of Acinetobacter calcoaceticus subsp. anitratus and subsp. lwoffi.