Gram-negative bacilli cause more than one third of all nosocomial infections in US hospitals. Despite a surfeit of new and highly potent antimicrobial agents, the problem of resistance in these pathogens continues to increase. Particularly important is the emergence of resistance to the fluoroquinolone and beta-lactam classes of antimicrobial agents. Recent work has confirmed that resistance to fluoroquinolone antibiotics is a complex process that involves mutations in the target enzymes (topoisomerase II and IV), decreased access to the target enzyme resulting from low permeability of the outer membrane (this is primarily important in Pseudomonas aeruginosa), and active efflux from the cell. Resistance to beta-lactam antibiotics, however, is primarily caused by the elaboration of an ever-growing number of beta-lactamases. Our ability to understand the genetic and biochemical underpinnings of these resistance phenotypes will be an important factor in determining the ultimate success of efforts to control their emergence and spread.