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Palmitoylation or S-acylation is the post-translational attachment of fatty acids to cysteine residues and is common among integral and peripheral mem brane proteins. Palmitoylated proteins have been found in every eukaryotic cell type examined (yeast, insect, and vertebrate cells), as well as in viruses grown in these cells. The exact functions of protein palmitoylation are not well understood. Intrin sically hydrophilic proteins, especially signaling molecules, are anchored by long chain fatty acids to the cytoplasmic face of the plasma membrane. Palmitoylation may also promote targeting to membrane subdomains enriched in glycosphingolip ids and cholesterol or affect protein-protein interactions. This chapter describes (1) a standard protocol for metabolic labeling of palmitoylated proteins and also the procedures to prove a covalent and ester-type linkage of the fatty acids, (2) a simple method to analyze the fatty acid content of S-acylated proteins, (3) two methods to analyze dynamic palmitoylation for a given protein and (4) protocolls to study cell-free palmitoylation of proteins.