Polymyxin-susceptible cells of Pseudomonas aeruginosa were exposed for 10, 30, and 60 min to growth medium containing 6000 units of polymyxin per millilitre. Exposure for 10 min resulted in lipid alterations in the cell envelope. A large reduction in the content of both phosphatidylethanolamine and phosphatidylglycerol with a large increase in both diphosphatidylglycerol and free fatty acids was found upon analysis by thin-layer chromatography. The cellular percentage of readily extractable lipid (REL) was reduced, and the phospholipid proportion of the REL decreased with polymyxin exposure. Polymyxin exposure for 30 and 60 min only slightly enhanced these chemical alterations. The cell envelope alterations found were characteristic for strains which become adaptively resistant to polymyxin. Treatment of the cells with chloramphenicol or KCN prior to polymyxin exposure did not prevent the lipid alterations from occurring. These observations suggest that the polymyxin-susceptible cell population adapts to polymyxin resistance by the rapid alteration of the cell envelopes of the entire cell population. We propose the theory that cell envelope phospholipases and proteases play a major role in the adaptive response and that the cation content of the cell envelope may be a critical controlling factor in the process.