Palmitoylation refers to the covalent attachment of a 16-carbon fatty acid to cysteine residues of proteins. This modification occurs on many intracellular signaling proteins including regulators of G protein signaling proteins (RGS). Palmitoylation mediates the interaction of proteins with membranes and other proteins and can control the biological activity of a protein. Palmitate attachment occurs through a labile thioester bond and is readily reversible in cells, thus providing a particularly important means for protein regulation. This chapter presents protocols for investigating RGS protein palmitoylation in mammalian cells. The RGS protein of interest is heterologously expressed in HEK293 cells, and cells are metabolically labeled with [3H]palmitate. The RGS protein is isolated from fractionated cells by immunoprecipitation and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography to determine if [3H] has been incorporated. To confirm that the radiolabeled fatty acid is linked to the protein through a thioester bond, labeled proteins are treated with neutral hydroxylamine. Oxyester-linked palmitate, which is occasionally found on serine and threonine residues, is insensitive to this treatment, whereas thioesters are sensitive. To verify that incorporated radiolabel is palmitate, the protein is treated with base, which also cleaves thioester bonds. The resulting lipids are extracted from the sample, then analyzed by chromatography.