The readily extractable lipids from a Pseudomonas aeruginosa isolate stepwse adapted to polymyxin resistance were compared with those of the susceptible parent and of a revertant strain which regained susceptibility. Significant qualitative and quantitative lipid alterations accompany the acquisition of resistance. Changes include the appearance of a major unidentified lipid (lipid X) unique to the readily extractable lipids of resistent cells. Comparative studies with parent and revertant strains indicated a significant decrease in the phospholipid content of resistant cells. Thin-layer chromatography of resistant-cell readily extractable lipids demonstrated: (i) the emergence of lipid X (36% of total readily extractable lipids), (ii) a decrease in phosphatidylethanolamine and phosphatidylglycerol, and (iii) an increase in diphosphatidylglycerol. Lipid X was purified by preparative silicic acid column chromatography and thin-layer chromatography and characterized by analytical thin-layer chromatography, column adsorption chromatography, and gas-liquid chromatography. Data from this study indicated that lipid X was a mixture of free fatty acids. The fatty acids present in lipid X were qualitatively and quantitatively the same as the fatty acids esterified to the phospholipids in the readily extractable lipids.