Acinetobacter baumannii is an opportunistic pathogen which has been implicated in a variety of nosocomial infections among immunocompromised patients worldwide. Recently, Multi-drug resistant (MDR) isolates of A. baumannii have been isolated from military personnel returning from service in Iraq and Afghanistan. Antibiotic resistance of A. baumannii has limited the number of active antibacterial, making very difficult to treat these types of infections. This work investigated the role of Resistance-Nodulation-cell Division (RND) efflux pumps in the antibiotic resistance mechanism of A. baumannii. Expression of six different RND pumps was analyzed in clinical isolates of A. baumannii. A novel RND family pump, AdeMNO, was found to be present in a majority of isolates. The adeMNO operon was cloned, sequenced, and characterized using the single copy gene expression system in an efflux sensitized surrogate Pseudomonas aeruginosa strain. Antibiotics, trimethoprim, chloramphenicol, and clindamicin were identified as the substrates of this pump. In order to understand the mechanisms of regulation of adeMNO operon, a putative regulator belonging to the lysR-family was identified, cloned, and sequenced from the upstream region of the operon. Promoter regions of the adeMNO operon were also sequenced from various clinical isolates and sequence polymorphisms identified that could be implicated in the regulation of adeMNO expression. / UOIT