The 72-kDa nuclear lamina protein lamin A is synthesized as a 74-kDa farnesylated precursor. Conversion of this precursor to mature lamin A appears to be mediated by a specific endoprotease. Prior studies of overexpressed wild-type and mutant lamin A proteins in cultured cells have indicated that the precursor possesses the typical carboxyl-terminal S-farnesylated, cysteine methyl ester and that farnesylation is required for endoproteolysis to occur. In this report, we describe the synthesis of an S-farnesyl, cysteinyl methyl ester peptide corresponding to the carboxyl-terminal 18 amino acid residues of human prelamin A. This peptide acts as a substrate for the prelamin A endoprotease in vitro, with cleavage of the synthetic peptide at the expected site between Tyr657 and Leu658. Endoproteolytic cleavage requires the S-prenylated cysteine methyl ester and, in agreement with transfection studies, is more active with the farnesylated than geranylgeranylated cysteinyl substrate. N-Acetyl farnesyl methyl cysteine is shown to be a noncompetitive inhibitor of the enzyme. Taken together, these observations suggest that there is a specific farnesyl binding site on the enzyme which is not at the active site.