Cell surface hydrophobicity properties vary dramatically, whereas cell envelope phospholipid composition is essentially identical among strains of Pasteurella multocida and Actinobacillus lignieresii. Fatty acid ester composition of the major phospholipid fractions from cell surface hydrophobicity variants was examined to determine whether hydrophobic properties are influenced by cell envelope fatty acid content. Individual phospholipids were resolved by preparative thin-layer chromatography, and methanolysis was performed with boron trifluoride-methanol. Gas-liquid chromatographic analysis revealed the organisms to be similar qualitatively, whereas hydrophobic variants exhibited consistently, greater and more disparate C16:0+C16:1/C14:0 ratios in all fractions. Fatty acid composition of phospholipids may be related to surface hydrophobicity properties of P. multocida variants. However, comparative data obtained for A. lignieresii revealed a degree of similarity with P. multocida that precludes use of this parameter as a means for differentiation of these Pasteurellaceae type species, thereby supporting their taxonomic relatedness.