Several studies have examined the role of palmitoylation of G protein alpha subunits by nonenzymatic in vitro acylation using palmitoyl-CoA. Here, we investigated nonenzymatic palmitoylation of purified G(s alpha) in vitro. GDP-bound G(s alpha) was stoichiometrically autoacylated on cysteine residue(s) with micromolar concentrations of palmitoyl-CoA The acylation led to a complete loss of steady-state GTPase activity and GTP gamma S binding to G(s alpha) Mutation of Cys 3 to Ala in G(s alpha) did not prevent either palmitoylation or its consequent functional alterations. However, stoichiometric palmitoylation of His(6)-G(s alpha) did not alter its GTPase activity or GTP gamma S binding. Isoelectric focusing of tryptic peptides from autoacylated wild type, His(6)-tagged, and C3A mutant of G(s alpha) showed that Cys 160 is the site of in vitro palmitoylation, Therefore, we conclude that in vitro palmitoylation of G(s alpha) occurs on Cys 160 and this modification decreases the ability of the protein to exchange GTP for GDP; N-terminus elongation of G(s alpha) prevents this latter effect without altering palmitoylation.