The rational design, synthesis, and activity of novel, hydroxamic acid-based, collective bisubstrate analog inhibitors of farnesyl protein transferase (FPT) is described. This class of compounds differ structurally from the conventional FPT inhibitors by being non-sulfhydryl and by being bisubstrate based rather than peptide or FPP derived inhibitors. Whereas replacement of the sulfhydryl group of tetrapeptide CVLS (I50 = 1 microM) by an N-methylhydroxamic acid had a deleterious effect (10, I50 > 360 microM), moderate inhibition was realized with 16 (I50 = 42.5 microM), a bisubstrate analog involving anchorage of farnesyl and tripeptide groups by a hydroxamic acid-embedded linker. Starting from 16, a 1 order of magnitude improvement in in vitro potency was obtained by optimization of the linker (20, I50 = 4.35 microM). An additional 13-fold enhancement was achieved by substituting the tripeptide moiety VLS in 20 by VVM (23, I50 = 0.33 microM). The dependence of these inhibitors on their peptide and farnesyl subunits is suggestive of their bisubstrate nature. Compound 23 (I50 = 0.33 microM) is 2 orders of magnitude better in activity compared to the initial lead 16 [I50 = 42.5 microM) and is effective in blocking prenylation of protein in whole cells including p21ras.