The aim of this study was to determine the epidemiological and molecular characteristics of hospital-acquired (HA)-methicillin-resistant Staphylococcus aureus (MRSA) isolates by investigating the distribution of clinical samples according to the hospital wards, antibiotic susceptibility patterns, staphylococcal chromosome cassette mec (SCCmec) types and the presence of Panton-Valentine Leukocidin (PVL) genes. A total of 110 MRSA isolates obtained from various clinical samples of inpatients at Hacettepe University Adult Hospital between January 2004 and December 2005 were included in the study. The identification of the isolates was done by BD Sceptor automated system (Becton Dickinson, USA). The mecA gene, SCCmec types and PVL genes were detected by polymerase chain reaction (PCR). Pulsed field gel electrophoresis (PFGE) was performed to examine the clonal relatedness. The susceptibility testing was performed for some antibiotics by E-test (AB Biodisk, Sweden) and for the others by disk diffusion methods according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. The clinical samples (35 blood, 37 pus, 23 deep tracheal aspiration, 5 catheter, and 10 other samples) that yielded the MRSA strains were isolated from patients (71.5%) at intensive care units and surgical wards. All the isolates were positive for mecA gene. Of the isolates, 68 (61.8%) were harboring SCCmec type III, 38 (34.5%) SCCmec variant IIIB, and 3 (2.7%) SCCmec type IV. One isolate which was mecA gene positive could not be classified in any of the SCCmec types. PVL was positive in 14 (12.7%) of the isolates. All MRSA strains were susceptible to tigecycline, linezolid, vancomycin and teicoplanin; however, exhibited high rates (> 90%) of resistance to gentamicin, ciprofloxacin and rifampin. Susceptibility rates to trimethoprim/sulfamethoxazole was 90%, clindamycin 53% and erythromycin 32%. Eight pulsotypes were distinguished on the basis of PFGE (A, B, C, D, K, L, N, O). Of the total isolates, 92.7% belonged to pulsotype A. HA-MRSA strains predominantly isolated from pus and blood samples of inpatients at intensive care units and surgical wards in our hospital were multi-resistant. Majority of these isolates were SCCmec III, or variant IIIB type. Although PVL is known as a common virulence factor of community-acquired MRSA, HA-MRSA isolates in our center have a considerable rate of PVL positivity pointing out the importance of surveillance of the changing epidemiology of MRSA.