Analogues of S‐prenylated cysteine like N‐acetyl‐S‐trans,trans‐farnesyl‐l‐cysteine (AFC) have previously been shown to inhibit the carboxyl methylation of proteins carrying a C‐terminal S‐prenylated cysteine residue and to block the endotoxin‐activated serum‐elicited ehemotactic response of mouse macrophages. Here, we show that AFC inhibits both basal and fonnyl peptide receptor‐stimulated binding of guanosine 5'‐O‐(3‐thiotriphosphate) (GTP[S]) to and hydrolysis of GTP by membranes of myeloid differentiated HL‐60 granulocytes. Receptor‐stimulated GTP[S] binding and GTP hydrolysis are more sensitive to AFC inhibition than basal G‐protein functions. Inhibition of fonnyl peptide receptor‐mediated G‐protein activation is also observed for S‐trans,trans‐famesyl‐3‐thiopropionic acid, but not for N‐acetyl‐S‐tarns‐geranyl‐l‐cysteine, N‐acetyl‐l‐cysteine, or the methyl ester of AFC, suggesting that the farnesyl moiety and the carboxyl group, but not the peptide bond of AFC are required for inhibition. The observations that exogeneous S‐adenosyl‐l‐methionine is apparently not required for and S‐adenosyl‐l‐homocysteine does not attenuate the inhibitory action of AFC raise the distinct possibility that AFC inhibits receptor‐mediated G‐protein interaction by a mechanism other than inhibition of protein carboxyl methylation.