Nitric oxide (NO) has emerged as one of several important intraovarian regulatory factors. In particular, NO has been implicated in the processes of ovulation and atresia-related apoptosis. The aim of the present study was to investigate the presence and distribution of the NO-generating nitric oxide synthase (NOS) enzymes in the ovary during follicular development, ovulation and luteal formation of the equine chorionic gonadotrophin (ECG)/human chorionic gonadotrophin (HCG)-primed rat. NADPH diaphorase activity was used as a histochemical marker for NOS within the ovary. Diaphorase reactivity was most abundant in the stroma (S) of the ovary and in the theca (T) layer of the follicle. In luteinized ovaries, weaker diaphorase reactivity was present within the corpora lutea (CL). Two different isoforms of NOS, the constitutively expressed endothelial NOS (eNOS) and the inducible isoform of NOS (iNOS), were immunolocalized in ovaries of immature rats and in ECG/HCG-primed rats during the periovulatory period from HCG injection until 2 days after ovulation. In addition, ovarian concentrations of eNOS and iNOS were quantified by immunoblotting. Immunoblotting with a monoclonal anti-eNOS antibody demonstrated the presence of eNOS mainly in the residual ovary (ROV) during the periovulatory period. In luteinized ovaries, higher concentrations of eNOS were seen in CL, while those in the ROV at this stage were lower than in the periovulatory ovary. Immature ovaries contained diminutive amounts of eNOS, detectable mostly in the ROV compartment. In contrast, iNOS was barely detectable during follicular development to the preovulatory stage. A slight elevation of iNOS was observed in the granulosa cells at 6 h after the HCG injection. The levels of iNOS during the luteal phase were also low. Immunohistochemical analysis using polyclonal eNOS and iNOS antibodies revealed the localization of these two isoforms primarily in the S and the T of the periovulatory ovary. In luteinized ovaries, positive immunoreactivity was also seen within the CL. With a monoclonal antibody against eNOS, intense immunoreactivity was observed in the S, T and within CL. There was a particularly strong staining in blood vessels. These data demonstrate the presence of an intraovarian NO-generating system. The localization of this system to the S, T and CL suggests a role for NO in the ovulatory process and in the regulation of CL function.