Acinetobacter baumannii is an opportunistic pathogen which is one of the most important agents of nosocomial infections. Being resistant to many antibiotics, complicates the treatment of the infections caused. Fluoroquinolones are used in the treatment of nosocomial and community-acquired infections and it is important to determine the mechanisms of resistance to these antibiotics. The aims of this study were to investigate ciprofloxacin (CIP) and levofloxacin (LEV) minimum inhibitor concentrations (MIC), clonal relationships, mutations that occur in DNA gyrase and topoisomerase IV genes and overexpression of efflux pumps in nosocomial A.baumannii isolates. A total of 81 A.baumannii strains, 79 CIP-resistant and two CIP-susceptible, isolated from different clinical samples in Ege University Faculty of Medicine, Department of Medical Microbiology were included in the study. CIP and LEV MIC values were determined by broth microdilution method. Clonal relationship among the strains was investigated by ERIC-PCR (Enterobacterial repetitive intergenic consensus-polymerase chain reaction). Mutations that occur in gyrA and parC genes were detected by DNA sequence analysis in 16 strains representing each clone and subtype. Overexpression of the efflux pumps was evaluated by broth microdilution and fluorometric accumulation experiments were carried out in the presence of pump inhibitors, among the representative strains. MIC50 and MIC90 values for CIP were 256 mu g/ml and >= 256 mu g/ml, while the values were 32 mu g/ml and 128 mu g/ml for LEV, respectively. Overall, CIP MIC values were found to be higher than that of LEV among the A.baumannii strains studied. Isolates were grouped into six main groups and 10 different clusters (39 strains in cluster A, 20 in B, 13 in C, two of each in I and J, one of each in D-H clusters), but it was observed that the majority of them were clonally related. The most common and important mutations detected in the gyrA and parC genes of the representative isolates were, Ser83Leu, Asp87Glu and Ser80Leu, respectively. While the overexpression of efflux pumps was observed in seven of the representative strains by microdilution method, in 11 by fluorometric assay and the results were positive in five of the strains by both methods. CIP was found to be better than LEV for detecting the overexpression of pumps by the use of microdilution method. It was concluded that, the reason of high fluoroquinolone MIC values of the studied nosocomial and clonally related A.baumannii strains were related to target mutations and overexpression of efflux pumps besides the plasmid-mediated mechanisms, such as Qnr might also have played a role.