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The aims of the present study were to determine: (1) whether oestradiol (E) in the superior mesenteric ganglion (SMG) modifies the release of ovarian progesterone (P), androstenedione (A) and E, the activity and gene expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 20α-HSD and the expression of P450 aromatase (Cyp19a1) and (2) whether any such modifications are related to changes in ovarian nitric oxide (NO) and noradrenaline (NA) levels during dioestrus II. Using an ex vivo SMG-ovarian nervous plexus-ovary system, ovarian P release was measured following the addition E plus tamoxifen (Txf) (10M) to the ganglion, whereas A, E, NA and NO were measured following the addition of E alone. Steroids were measured by radioimmunoassay, NA concentrations were determined by HPLC and gene expression was evaluated using reverse transcription-polymerase chain reaction. Oestradiol in the ganglion decreased ovarian P, E and NA release, as well as 3β-HSD activity, but increased the release of A and nitrites, as well as the 20α-HSD expression and its activity. No changes were observed in Cyp19a1 gene expression. The addition of E plus Txf to the ganglion reversed the effects of E alone. The action of oestradiol in SMG favours the beginning of functional luteolysis, due to an increase in NO release and a decrease in NA in the ovary. These results may help elucidate the role of E in hormone-dependent pathologies in women.