It has recently been reported that a precursor of p21ras (pro-p21ras) becomes modified by a metabolite of mevalonic acid prior to conversion to mature p21ras. We have examined the effect of blocking isoprenoid biosynthesis on this process. Fluoromevalonate, which inhibits the conversion of pyrophosphomevalonate to isopentenyl pyrophosphate, blocks the incorporation of radioactive mevalonate into pro-p21ras, demonstrating the mevalonate must be converted to an isoprenoid prior to such incorporation. Starvation of CHO-K1 cells for mevalonic acid by treatment with mevinolin, an inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, or mevalonate deprivation in a mevalonate auxotroph defective in HMG-CoA synthase activity results in the accumulation of pro-p21ras. The precursor, accumulated due to either of these treatments, is converted through an intermediate form to the mature p21ras by incubation of cells with mevalonate. Incubation of cells with 25-hydroxycholesterol, the pleiotropic transcriptional down-regulator of cholesterol biosynthesis does not, however, result in the accumulation of pro-p21ras. This result indicates that in contrast to the regulation of cholesterol biosynthesis in mammalian cells, important regulatory control other than at the level of HMG-CoA reductase is involved in the isoprenoid biosynthesis required for protein isoprenylation.