The type strain and several clinical isolates of Corynebacterium amycolatum were examined for lipid composition as a chemotaxonomic character for routine identification. The phospholipid profile was composed of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides, together with various unidentified compounds. One of them, accounting for 20-29% of total phospholipids, was purified and characterized as acyl phosphatidylglycerol by chromatographic and spectrometric techniques. The acyl substituents on the phosphatidyl moiety were characterized as tetradecanoyl, pentadecanoyl, hexadecenoyl, hexadecanoyl, heptadecenoyl, heptadecanoyl, octadecenoyl (the major one), and octadecanoyl. The acyl group on the polar head (glycerol) was only octadecenoyl. Phospholipid analysis by thin-layer chromatography of a collection of Corynebacterium strains proved that this compound is widely distributed, although it only represents a minor (2-9%) component among mycolic acid-containing species. Acyl phosphatidylglycerol can be considered as a useful chemical marker for the identification of C. amycolatum in addition to the absence of mycolic acids.