The antiproliferative effect of osthole on rat vascular smooth muscle cells was examined in this study. A number of mitogenic agents, e.g., foetal-calf serum (10%, v/v) and platelet-derived growth factor (20 ng/ml), and pharmacological agents, e.g., serotonin (10 μM), ionomycin (3 nM), phorbol 12,13-dibutyrate (20 nM) and phorbol myristate acetate (200 nM), were used to induce DNA synthesis in rat vascular smooth muscle cells; these effects were concentration dependently inhibited by osthole and the half-maximal inhibition (IC 50) occurred at 13.6 ± 1.8, 11.8 ± 1.3, 7.9 ± 0.9, 7.1 ± 0.2, 7.8 ± 0.2 and 8.6 ± 0.4 μM, respectively. Osthole itself increased the cyclic AMP and cyclic GMP formations in a concentration-dependent manner; it synergistically increased cyclic AMP and cyclic GMP levels induced by forskolin and sodium nitroprusside, respectively. After 48 h deprivation of serum, cells were re-stimulated with serum and the cell cycle was observed by flow cytometry; treatment of cells with osthole (100 μM) caused a block of serum-inducible cell cycle progression at a point before the G 1- S boundary. The addition of osthole (100 μM) at various times after serum addition to serum-deprived cells showed full inhibition of DNA synthesis even when added 6 h after serum. The cell cycle progression block was gradually lost as the delay from serum to osthole application was increased from 6 to 18 h. The effect of osthole on serum-stimulated [ 3H]thymidine incorporation into endothelial cells was examined and the IC 50 value (158.7 ± 2.7 μM, n = 6) was obtained; it exhibited greater potency (12-fold) for vascular smooth muscle cells as compared with endothelial cells as an antiproliferative agent. These results suggest that osthole is a selective antiproliferative agent in vascular smooth muscle cells. The antiproliferative effect occurs at the early G 1 phase of the cell cycle and is due to the increase in cyclic AMP and cyclic GMP contents.