Several proteins in mammalian cells are modified post-translationally by the isoprenoid, farnesol. Incubation of cultured cells with [3H]mevalonate, an isoprenoid precursor, results in the labeling of multiple polypeptides, the most prominent of which migrate in the range of 21-26 kDa on sodium dodecyl sulfate-polyacrylamide gels. In Rat-6 fibroblasts transformed by H-ras, one of the farnesylated proteins was identified as p21ras by two-dimensional immunoblotting. However, this protein accounted for only a small proportion of the [3H]mevalonate-derived radioactivity incorporated into 21-26-kDa proteins. Murine erythroleukemia cells, which did not express immunodetectable quantities of p21ras, contained several 21-26-kDa farnesylated proteins distributed in both the cytosolic and particulate fractions. At least eight of these proteins were capable of binding [alpha-32P]GTP on nitrocellulose membranes. Pulse-chase studies showed that the isoprenoid modification did not necessarily result in the translocation of the cytosolic proteins to the cell membrane. A prominent group of carboxyl-methylated proteins in murine erythroleukemia cells overlapped with the 21-26-kDa farnesylated proteins on one-dimensional sodium dodecyl sulfate gels. Methylation of this group of proteins was selectively abolished when cells were treated with lovastatin, an inhibitor of isoprenoid synthesis. Addition of exogenous mevalonate to the lovastatin-treated cells fully restored carboxyl methylation. These studies suggest that the 21-26-kDa farnesylated proteins in mammalian cells are members of a recently discovered family of low molecular mass GTP-binding proteins which, although ras-related, appear to be distinct structurally and possibly functionally from the products of the ras genes. The observed isoprenoid-dependent carboxyl methylation of a group of 21-26-kDa proteins suggests that the low molecular mass GTP-binding proteins may undergo a series of post-translational C-terminal cysteine modifications (i.e. farnesylation, carboxyl methylation) analogous to those recently elucidated for p21ras.