Nitric oxide (NO) has recently emerged as a potential regulator of follicular development because of its involvement in the regulation of several physiological functions of the ovary. NO influences apoptotic cell death of follicular cells as a follicle survival factor. The present study was conducted (1) to investigate the mechanism involved in the protective effect of NO on spontaneously induced follicular apoptosis in serum-free condition and (2) to determine the role of NO on the expression of mRNAs and proteins for HSP70 and Bax. Preovulatory follicles obtained from PMSG-primed rats were cultured for 24 hr in serum-free medium with or without sodium nitroprusside (SNP), a NO generator. Granulosa cells within follicles incubated in medium alone for 24 hr exhibited extensive apoptosis. Treatment of SNP in the culture medium blocked this onset of apoptosis. Both mRNA and protein levels of HSP70 were highly increased with SNP than those of control group. On the contrary, those of Bax were suppressed with SNP treatment. Results of the present study suggest that NO prevents rat preovulatory follicular apoptosis in vitro by stimulating HSP70 and suppressing Bax expression.