Members of the Ras superfamily of small GTP-binding proteins, γ-subunits of heterotrimeric G proteins and nuclear lamin B are subject to a series of post-translational modifications that produce prenylcysteine methylester groups at their carboxyl termini. The thioether-linked polyisoprenoid substituent can be either farnesyl (C 1 5 ) or geranylgeranyl (C 2 0 ). Small molecule prenylcysteine derivatives with either the C 1 5 or C 2 0 modification, such as N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), S-trans,trans-farnesylthiopropionate (FTP), as well as the corresponding geranylgeranyl derivatives (AGGC and GGTP) are substrates for the carboxyl methyltransferase. Saccharomyces cerevisiae ste 14 mutants that lack RAS and a-factor carboxyl methyltransferase activity are also unable to methylate farnesyl and geranylgeranylcysteine derivatives. Moreover, C 2 0 -substituted cysteine analogs directly compete for carboxyl methylation with the C 1 5 -substituted cysteine analogs and vice versa. Finally, AGGC is even more effective than AFC as an inhibitor of Ras carboxyl methylation, despite the fact that Ras is methylated at a farnesylcysteine rather than a geranylgeranylcysteine residue.