AbstractThe article summarises the results of more than 30 years of research on palmitoylation (S‐acylation) of viral proteins, the post‐translational attachment of fatty acids to cysteine residues of integral and peripheral membrane proteins. Analysing viral proteins is not only important to characterise the cellular pathogens but also instrumental to decipher the palmitoylation machinery of cells. This comprehensive review describes methods to identify S‐acylated proteins and covers the fundamental biochemistry of palmitoylation: the location of palmitoylation sites in viral proteins, the fatty acid species found in S‐acylated proteins, the intracellular site of palmitoylation and the enzymology of the reaction. Finally, the functional consequences of palmitoylation are discussed regarding binding of proteins to membranes or membrane rafts, entry of enveloped viruses into target cells by spike‐mediated membrane fusion as well as assembly and release of virus particles from infected cells. The topics are described mainly for palmitoylated proteins of influenza virus, but proteins of other important pathogens, such as the causative agents of AIDS and severe acute respiratory syndrome, and of model viruses are discussed.