Burkholderia multivorans causes opportunistic pulmonary infections in cystic fibrosis and immunocompromised patients. The purpose of the present study was to determine the nature of the phospholipids and their fatty acid constituents comprising the cell envelope membranes of strains isolated from three disparate sources. A conventional method for obtaining the readily extractable lipids fraction from bacteria was employed to obtain membrane lipids for thin-layer chromatographic and gas chromatography-mass spectrophotometric analyses. Major fatty acid components of the B. multivorans readily extractable lipid fractions included C16:0 (palmitic acid), C16:1 (palmitoleic acid), and C18:1 (oleic acid), while C14:0 (myristic acid), ΔC17:0 (methylene hexadecanoic acid), C 18:0 (stearic acid), and ΔC19:0 (methylene octadecanoic acid) were present in lesser amounts. Fatty acid composition differed quantitatively among strains with regard to C16:0, C 16:1, ΔC17:0, C18:1, and ΔC 19:0 with the unsaturated:saturated fatty acid ratios being significantly less in a cystic fibrosis type strain than either environmental or chronic granulomatous disease strains. Phospholipids identified in all B. multivorans strains included lyso-phosphatidylethanolamine, phosphatidylglycerol, phosphatidylethanolamine, and diphosphatidylglycerol in similar ratios. These data support the conclusion that the cell envelope phospholipid profiles of disparate B. multivorans strains are similar, while their respective fatty acyl substituent profiles differ quantitatively under identical cultivation conditions.