Acinetobacter baumannii, unnamed Acinetobacter species 3 (studied by P.J.M. Bouvet and P.A.D. Grimont) and unnamed DNA group 13 (studied by I. Tjernberg and J. Ursing) are the most prevalent Acinetobacter species in hospitals. Using the identification scheme of Bouvet and Grimont, it is sometimes difficult to differentiate these species from A. calcoaceticus sensu stricto, a species of the natural environment that has seldom been found associated with human infection. Genetically identified Acinetobacter isolates belonging to A. calcoaceticus sensu stricto (n = 12), A. baumannii (n = 22), Acinetobacter species 3 (n = 15) and DNA group 13 of Tjernberg and Ursing (n = 26), Acinetobacter species 10 (n = 2), Acinetobacter species 11 (n = 2) and 3 strains ungrouped by DNA-DNA hybridization were investigated for electrophoretic separations of L-malate dehydrogenase (MDH), glutamate dehydrogenase (GDH) and catalase (CAT). All A. calcoaceticus sensu stricto isolates were easily differentiated from those of other species investigated by their high MDH values (relative mobility (Rf) = 78), their low GDH values (Rf range: 24-28) and CAT values (Rf range: 34-42). Acinetobacter species 3 was differentiated from A. baumannii and DNA group 13 of Tjernberg and Ursing by high CAT values. A. baumannii could not be differentiated from Tjernberg and Ursing DNA group 13. Acinetobacter species 10 was clearly differentiated from Acinetobacter species 11. Once an Acinetobacter is phenotypically identified with the four closely related species investigated here, electrophoretic analysis of MDH, GDH and CAT might be a useful complement to the identification scheme of Bouvet and Grimont for accurately identifying A. calcoaceticus sensu stricto.