The enzyme protein-farnesyl transferase (PFTase) catalyzes the farnesylation of the Ras protein and certain other proteins, using farnesyl diphosphate (FPP) as the prenyl source. Because of the important role of mutant Ras proteins in cancer, inhibitors of PFTase are of great interest as potential novel anticancer agents. The design of such agents would be aided by a greater knowledge of the mechanism of PFTase. We have determined the stereochemical course of PFTase using the two stereospecifically prepared isomers of [1-H-2]-FPP as substrates in conjunction with H-1-NMR analysis of the farnesylated peptide products. This confirms that PFTase carries out the transfer of the farnesyl group with inversion of configuration. Combined with the results of studies on fluorinated analogs of FPP as PFTase substrates (Dolence, J. M.; Poulter, C. D. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 5008), this indicates that the mechanistic course of PFTase is similar to that of the prototypical prenyltransferase enzyme, FPP synthase.