Resistance to carbapenems in Acinetobacter baumannii is increasingly reported and is mostly associated with expression of carbapenemases, most frequently carbapenem-hydrolyzing class D β-lactamases. Four major subgroups exist in A. baumannii: naturally occurring OXA-51/69-type and acquired OXA-23, OXA-24/40, OXA-58-type β-lactamases. OXA-51/69 might contribute to resistance via ISAba1 insertion in its promoter region. Twenty nonrepetitive carbapenem-resistant A. baumannii clinical isolates were studied. All were resistant to imipenem (8 additionally to meropenem, MICs 8-64 μg/ml). No metallo-β-lactamase production was detected. PFGE revealed 5 distinct clones (I-V). PCR showed 16 isolates (clones I-III) had blaOXA-58; none had blaOXA-23/40; all had blaOXA-51. In isolate Ab5, ISAba1 upstream of blaOXA-51 was truncated by novel insertion sequence ISAba9 (974 bp, IS982 family, 17-bp inverted repeats, 8-bp target site duplication TTGTTTAA). A hybrid promoter (35 box in ISAba9, 10 box in ISAba1, optimal 17-bp spacing) was identified. Quantitative RT-PCR showed 8-fold higher blaOXA-51 expression in Ab5 (ISAba1+ISAba9) vs clonally related Ab2 (ISAba1 alone). PCR screening found ISAba9 in 4 isolates but only upstream of blaOXA-51 in Ab5. In conclusion, novel ISAba9 is involved in blaOXA-51 gene overexpression, contributing to carbapenem resistance in A. baumannii.