Acinetobacter has been reported to be involved in hospital-acquired infections with increasing frequency. However, clinical laboratories still lack simple methods that allow the accurate identification of Acinetobacter strains at the species level. For this study, proteinase K-digested whole-cell lysates from 44 clinical and environmental isolates were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with hyperimmune rabbit sera to examine the possibility of developing a serotyping scheme based on the O antigen of Acinetobacter lipopolysaccharide (LPS). The antisera, obtained by immunization of rabbits with 13 of the heat-killed isolates investigated, were characterized by Western blotting and enzyme immunoassay by using proteinase K-digested whole-cell lysates and phenol-water-extracted LPS as antigens. In both assays, the antisera were shown to be highly specific for the homologous antigen. In addition, assignment of Acinetobacter LPS to the smooth or the rough phenotype was shown not to be reliable when it was based only on the results obtained with silver-stained gels. O-antigen reactivity, determined by Western blot analysis, was observed with 11 of the 31 isolates, most of which belonged to the species Acinetobacter baumannii (DNA group 2) and the unnamed DNA group 3. Interestingly, some O antigens were found in a DNA group different from that of the strain used for immunization. The results indicate that O serotyping of Acinetobacter strains is feasible and thus may provide a simple method for the routine identification of these opportunistic pathogens.