We characterized two new gene cassettes in an Acinetobacter isolate: one harbored the metallo-β-lactamase (IMP-4) gene bla_IMP-4, the other harbored the rifampin ADP-ribosyltransferase (ARR-2) gene arr-2, and both arrayed with the aminoglycoside acetyltransferase [AAC(6′)-Ib₇] gene cassette aacA4 in two separate class 1 integrons. The epidemiology of these gene cassettes in isolates from blood cultures obtained from 1997 to 2000 was studied. Isolates bearing either the bla_IMP-4 or the arr-2 gene cassette or both represented 17.5% (10 of 57) of isolates in 1997, 16.1% (10 of 62) in 1998, 2.5% (1 of 40) in 1999, and 0% (0 of 58) in 2000. These two gene cassettes, probably borne on two separate integrons, were found in at least three genomic DNA groups, with evidence of clonal dissemination in the intensive care unit during 1997 to 1998. Seventeen of the 52 Acinetobacter baumannii (genomic DNA group 2) isolates from 1997 to 2000 harbored intI1, but only one was positive for these gene cassettes, whereas 20 of the 21 intI1-positive isolates of all other genomic DNA groups were positive for either or both of them. Reduced susceptibility to imipenem and rifampin was seen only in isolates harboring the bla_IMP-4 and arr-2 cassettes, respectively. The aminoglycoside phosphotransferase [APH(3′)-VIa] gene aph(3′)-VIa was detected in all 21 isolates for which the MIC of amikacin was ≥8 μg/ml, with or without aacA4, whereas aacA4 alone was found in isolates for which the MIC of amikacin was 0.5 to 2 μg/ml. Significant differences between the 17 intI1-positive and 47 intI1-negative isolates belonging to genomic DNA group 3 from 1997 to 1998 in the MICs of amikacin, gentamicin, imipenem, sulfamethoxazole, and ceftazidime were observed (Mann-Whitney test, P < 0.001 to 0.01).