To evaluate the effect of nitric oxide (NO.) in human corpus luteum (CL) function, we investigated the expression and the presence of NO. synthase (NOS) in the human CL and the action of NO. on the in vitro luteal steroid production. The expression of endothelial NOS (eNOS) in early, mid-, and late CL was assessed by reverse transcriptase polymerase chain reaction (RT-PCR) and the immunohistochemical study was performed in human CL histological sections by using monoclonal antibodies (MAbs) against the distinct NOS isoforms. In addition, seven human mid-CLs were enzymatically dispersed, and the cells were cultured with NO. donor compounds. Steroid production was measured in the culture media by specific radioimmunoassay. The results show that the expression of eNOS was highly detected in mid- and early CL, and to a lesser extent, in late CL. Meanwhile, the immunohistochemical study indicated that both isoenzymes of NOS were expressed in mid-human CL, eNOS being the more abundant isoform present. On the other hand, functional studies showed that NO. donors (L-arginine [L-Arg] and sodium nitroprusside) elicited an inhibitory action on steroid synthesis, preferentially on estradiol production by the luteal cell cultures (p < 0.05). In conclusion, the NO-NOS system is present in the human CL, and it may serve as a modulator of the in vitro human luteal steroidogenesis.