The mechanism of colistin resistance (Col(r)) in Acinetobacter baumannii was studied by selecting in vitro Col(r) derivatives of the multidrug-resistant A. baumannii isolate AB0057 and the drug-susceptible strain ATCC 17978, using escalating concentrations of colistin in liquid culture. DNA sequencing identified mutations in genes encoding the two-component system proteins PmrA and/or PmrB in each strain and in a Col(r) clinical isolate. A colistin-susceptible revertant of one Col(r) mutant strain, obtained following serial passage in the absence of colistin selection, carried a partial deletion of pmrB. Growth of AB0057 and ATCC 17978 at pH 5.5 increased the colistin MIC and conferred protection from killing by colistin in a 1-hour survival assay. Growth in ferric chloride [Fe(III)] conferred a small protective effect. Expression of pmrA was increased in Col(r) mutants, but not at a low pH, suggesting that additional regulatory factors remain to be discovered.