We report the ability of Giardia lamblia to modify several of its cellular proteins by isoprenylation. Trophozoites cultured in the presence of [3H]mevalonate synthesized radiolabeled proteins of approx. 50 and 21-26 kDa. Chemical analysis indicated that farnesyl and geranylgeranyl isoprenoids comprised the majority of the radiolabel covalently associated with trophozoite proteins. In addition, antibodies to human p21ras immunoprecipitated mevalonate-labelled species of approx. 21 kDa. Inhibitors of several enzymatic steps of the mevalonate pathway dramatically affected Giardia metabolism. Protein isoprenylation and cell growth were blocked by compactin and mevinolin, competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme in isoprenoid biosynthesis. In the presence of these inhibitors, Giardia growth was restored by the addition of mevalonate to the culture medium. In contrast, cell growth was blocked irreversibly by inhibitors of subsequent steps in the protein isoprenylation pathway. Trophozoite growth inhibition by limonene, perillic acid, perillyl alcohol and N-acetyl-S-farnesyl-L-cysteine was not reversed after the addition of mevalonate, dolichol, ubiquinone or cholesterol to the medium. These observations constitute the first description of protein isoprenylation in any protozoan and indicate that this post-translational modification is an important step in the regulation of the growth of this primitive eukaryote.