Acinetobacter calcoaceticus strain BM2500 was resistant to ampicillin, aminoglycoside-aminocyclitols, chloramphenicol, sulfonamides, and high levels of trimethoprim. Resistance to ampicillin was due to the presence of a beta-lactamase (TEM-1) and the aminoglycoside-aminocyclitol resistance was mediated by phosphotransferase (APH(3')(5")I) and adenylyltransferase (AAD(3)(9] activities. The resistance genes were carried by a 167 kilobase plasmid, pIP1031, belonging to incompatibility group 6-C; the plasmid was self-transferable, at extremely low frequency, to Escherichia coli by conjugation. Plasmid pIP1031 DNA was analyzed by agarose gel electrophoresis following restriction endonuclease digestion, by nucleic acid hybridization, and by CsCl analytical density gradient ultracentrifugation. The results support the hypothesis that plasmid pIP1031 may have been acquired recently by strain BM2500.