S-diclofenac (2-[(2,6-dichlorophenyl) amino] benzene acetic acid 4-(3H-1,2,dithiol-3-thione-5-yl) phenyl ester) is a novel molecule comprising a hydrogen sulfide (H2S)-releasing dithiol-thione moiety attached by an ester linkage to diclofenac. Effect of S-diclofenac (H2S donor) on cell proliferation was investigated on the primary and immortalized rat aortic vascular smooth muscle cells (SMC). Smooth muscle cell proliferation has been considered as a key event in vascular injury in diseases such as atherosclerosis and restenosis after invasive intervention. Clonogenic cell survival assay showed a dose dependent (10-100 microM) decrease in cell survival. Flow cytometric analysis showed that the asynchronized cells are more sensitive than the cells that are synchronized and revealed that the cells in G1 phase are not affected by the treatment of the S-diclofenac. Asynchronized smooth muscle cells treated with the S-diclofenac showed an increase in apoptotic cell death. S-diclofenac treatment also resulted in stabilization of p53 coupled with the induction of downstream proteins such as p21, p53AIP1 and Bax. S-diclofenac did not up-regulate cell levels of the antiapoptotic protein Bcl-2. However, when the cells are synchronized a stimulatory effect of cell growth with the decrease in apoptosis, p53 and p21 was evident. S-diclofenac inhibits smooth muscle cell growth and may play a role in the lesion formation at sites of the vascular injury. The present results suggest that S-diclofenac may be useful for the prevention of smooth muscle cell proliferation in diseases such as vascular obstructive and restenosis.