Recent evidence suggests that apoptosis may be involved in the control of vascular smooth muscle cell (VSMC) number in atherosclerotic lesions. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been reported to induce apoptosis in a variety of tumor cell lines. To evaluate whether these agents also induce apoptosis of VSMCs, cultured rat VSMCs were treated with increasing doses of atorvastatin in the presence of FBS as a survival factor. The presence of apoptosis was evaluated by morphological criteria, annexin V binding, and DNA fragmentation and quantified as the proportion of hypodiploid cells by flow cytometry. Atorvastatin induced apoptosis in a dose-dependent manner, an effect also seen with simvastatin and lovastatin, but not with the hydrophilic drug pravastatin. The proapoptotic effect of statins was seen only when the inhibition of acetate incorporation into sterols was >95% and was fully reversed by mevalonate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate but not by isopentenyl adenosine, ubiquinone, or squalene, suggesting a role for prenylated proteins in the regulation of VSMC apoptosis. To further assess the role of protein prenylation, VSMCs were exposed to the prenyl transferase inhibitors perillic acid and manumycin A. Both agents induced VSMC apoptosis as evaluated by the above-mentioned criteria. Finally, VSMC treatment with lipophilic statins was associated with decreased prenylation of p21-Rho B, further supporting the role of protein prenylation inhibition in statin-induced VSMC apoptosis. The present data suggest that interference with protein prenylation by HMG-CoA reductase inhibitors or other agents may provide new strategies for the prevention of neointimal thickening.