The unambiguous identification of Acinetobacter strains, particularly those belonging to the Acinetobacter calcoaceticus-Acinetobacter baumannii complex, is often hindered by their close geno- and phenotypic relationships. In this study, monoclonal antibodies (MAbs) against the O antigens of the lipopolysaccharides from strains belonging to the A. calcoaceticus-A. baumannii complex were generated after the immunization of mice with heat-killed bacteria and shown by enzyme immunoassays and Western blotting to be specific for their homologous antigens. Since the A. calcoaceticus-A. baumannii complex comprises the most clinically relevant species, the MAbs were subsequently tested in dot and Western blots with proteinase K-treated lysates from a large collection of Acinetobacter isolates (n = 631) to determine whether the antibodies could be used for the reliable identification of strains from this complex. Reactivity was observed with 273 of the 504 isolates (54%) from the A. calcoaceticus-A. baumannii complex which were included in this study. Isolates which reacted positively did so with only one antibody; no reactivity was observed with isolates not belonging to the A. calcoaceticus-A. baumannii complex (n = 127). To identify additional putative O serotypes, isolates from the A. calcoaceticus-A. baumannii complex which showed no MAb reactivity were subjected to a method that enables the detection of lipid A moieties in lipopolysaccharides with a specific MAb on Western blots following acidic treatment of the membrane. By this method, additional serotypes were indeed identified, thus indicating which strains to select for future immunizations. This study contributes to the completion of a serotype-based identification scheme for Acinetobacter species, in particular, those which are presently of the most clinical importance.