Covalently linked fatty acids are increasingly recognized as an important type of post-translational protein modification. Many of such acylated proteins are found associated with cellular membranes. The membrane skeleton of the parasitic hemoflagellate Trypanosoma brucei consists of a regular array of microtubules which are tightly bound to the overlying cell membrane. A microtubule-binding protein (p41) has been identified within this structure which carries covalently bound fatty acid. The fatty acid linkage is sensitive to hydroxylamine treatment. After chemical transesterification, the released radioactivity co-migrates with fatty acid methyl esters in thin-layer chromatograms, establishing that the fatty acid was covalently bound to p41 via an ester (thioester) linkage. Upon detergent extraction of trypanosomes, p41 remains tightly bound to the cytoskeleton as long as Ca2+ ions are present. It can selectively be released from this structure by the addition of excess EGTA. Conversely, p41 binds to isolated cytoskeletons and to purified microtubules in vitro, the reaction again being entirely Ca2+-dependent.