Acinetobacter baumannii strains resistant to all beta-lactams, aminoglycosides, and fluoroquinolones have emerged in many medical centers. Potential mechanisms contributing to antimicrobial resistance were investigated in 40 clinical isolates endemic to New York City. The isolates were examined for the presence of various beta-lactamases, aminoglycoside-modifying enzymes, and mutations in gyrA and parC. Expression of the genes encoding the beta-lactamase AmpC, the efflux systems AdeABC and AbeM, and the OmpA-like porin was also examined by real-time reverse transcription-PCR. No VIM, IMP, KPC, OXA-23-type, OXA-24-type, or OXA-58 beta-lactamases were detected, although several isolates had acquired bla(SHV-5). Most cephalosporin-resistant isolates had increased levels of expression of ampC and/or had acquired bla(SHV-5); however, isolates without these features still had reduced susceptibility to cefepime that was mediated by the AdeABC efflux system. Although most isolates with ISAba1 upstream of the bla(OXA-51)-like carbapenemase gene were resistant to meropenem, several remained susceptible to imipenem. The presence of aminoglycoside-modifying enzymes and gyrase mutations accounted for aminoglycoside and fluoroquinolone resistance, respectively. The increased expression of adeABC was not an important contributor to aminoglycoside or fluoroquinolone resistance but did correlate with reduced susceptibility to tigecycline. The expression of abeM and ompA and phenotypic changes in OmpA did not correlate with antimicrobial resistance. A. baumannii has become a well-equipped nosocomial pathogen; defining the relative contribution of these and other mechanisms of antimicrobial resistance will require further investigation.