Cysteine farnesylation of the ras oncogene product, p21ras, on its carboxyl-terminal CA1A2X box (C = cysteine, A = aliphatic, and X = methionine or serine) is required for its transforming activity. p21ras farnesyltransferase (FTase), the enzyme responsible or this important posttranslational modification can be inhibited by simple CA1A2X peptides. We have synthesized a family of CA1A2X peptidomimetics where the central 2 aliphatic amino acids are replaced by a variety of spacer groups with different shapes and conformational characteristics to investigate the structural requirements of these inhibitors. The biological activities of CA1A2X peptidomimetics, where the dipeptide "A1A2" is replaced by 3- or 4-aminomethylbenzoic acid (AMBA) and 3- or 4-aminobenzoic acid (ABA), are evaluated in a p21ras FTase inhibitory assay. Peptidomimetics Cys-4-ABA-Met and Cys-3-AMBA-Met contain spacers that provide good distance correspondence of the carboxylate and ammonium separation with that of the parent KB p21ras tetrapeptide, Cys-Val-Ile-Met, and are as potent FTase inhibitors (IC50 values of 50 and 100 nM, respectively). In contrast, replacing the central dipeptide with 4-AMBA reduced FTase inhibitory activity by 17-fold whereas replacement by 3-ABA reduces inhibitory activity of the peptidomimetics by 43-fold. Cys-4-ABA-Met (IC50 = 50 nM) is 128 times more potent as a p21ras FTase inhibitor than Cys-3-ABA-Met (IC50 = 6400 nM), yet these two peptidomimetics differ only in the substitution pattern around the phenyl ring. These results coupled with computer modeling studies demonstrate that the interaction between FTase and the peptidomimetics requires precise structural and conformational characteristics; in particular, correct positioning of the Cys and Met must be respected. Furthermore, Cys-3-AMBA-Met and Cys-4-ABA-Met are true inhibitors of p21ras FTase since they are not farnesylated by this enzyme, in contrast to Cys-Val-Ile-Met, which inhibits the enzyme by acting as alternative substrate. Computer modeling studies of the potent FTase inhibitor Cys-4-ABA-Met show that a folded conformation, where the thiol and carboxylate groups are close, is not possible. Therefore a beta-turn conformation that would result in the simultaneous coordination of the Cys-thiol and Met-carboxylate to zinc ion is not important for inhibition of p21ras FTase, as previously suggested.