The phospholipid class and molecular species compositions of bacteriophage phi6 and its host Pseudomonas syringae were determined quantitatively using TLC and liquid-chromatography/electrospray ionization mass-spectrometry. In addition, the fatty acid compositions of the phospholipids were analyzed by gas-chromatography/mass-spectrometry. The phage contained significantly more phosphatidylglycerol (PG) and less phosphatidylethanolamine (PE) than the host cytoplasmic (CM) and outer (OM) membranes. In addition, the phospholipid molecular species composition of the viral membrane differed from those of the host membranes, but resembled that of CM more than OM as shown by principal component analysis (PCA). The membrane of phi6 contained more 34:1 and 34:2, and less 32:1 PE and PG molecular species than the host CM or OM. Also, phi6 contained negligible amounts of saturated phospholipid molecular species. These data provide the first biochemical evidence suggesting that phi6 obtains its lipids from the CM. This process is not unselective, but certain phospholipid species are preferentially incorporated in the phage membrane. Common factors leading to similar enrichment of PG in every membrane-containing bacterial virus system studied so far (phi6, PM2, PRD1, PR4, Bam35) are discussed.