Ras proteins are mutationally activated in a variety of human cancers. Since farnesylation of Ras proteins is required for expression of their oncogenic potential, the enzyme responsible for this reaction, farnesyl:protein transferase (FPT), has become a major target for anticancer drug development. FPT is a zinc metalloenzyme, and the zinc is essential for its catalytic activity. To begin to elucidate the role of zinc in catalysis, we initiated metal substitution studies. Of all metals tested, only cadmium was able to functionally substitute for zinc, reconstituting enzymatic activity with native substrates (H-Ras and farnesyl diphosphate) to about 50% of that of the zinc-containing enzyme. Several important differences were observed between cadmium-substituted FPT (Cd-FPT) and zinc-containing FPT (Zn-FPT). Cd-FPT not only uses H-ras with its native CaaX motif (Ras-CVLS) as a substrate but also can farnesylate H-ras in which the CaaX motif is altered to contain a C-terminal leucine residue (Ras-CVLL). Similarly, Cd-FPT can farnesylate leucine-terminated peptides. Leucine-terminated proteins and peptides are usually substrates for the related enzyme geranylgeranyl:protein transferase type I. Farnesylation of Ras-CVLS and Ras-CVLL by Cd-FPT exhibited similar sensitivity to the FPT inhibitor SCH 44342 and to the peptide inhibitor CAIM. However, unlike Zn-FPT, Cd-FPT is also potently inhibited by the leucine-terminated peptide CAIL. These results indicate that the metal ion content of FPT strongly influences its protein substrate specificity.