The M2 protein of influenza A virus, a 97 amino acid integral membrane protein expressed on the surface of infected cells, is covalently modified with long chain fatty acids. The fatty acid bond is sensitive to treatment with neutral hydroxylamine and mercaptoethanol, which indicates a labile thioester type linkage. Thin-layer chromatographic fatty acid analysis of [3H]myristic and [3H]palmitic acid-labelled M2 protein shows that palmitic acid is the predominant fatty acid linked to this polypeptide. Palmitoylation of M2 occurs post-translationally and causes an upward shift in the SDS-PAGE mobility of the protein.