The rat posterodorsal medial amygdala (MePD) is a sexually dimorphic area implicated in the control of reproduction. Interestingly, nitric oxide (NO) synthetizing neurons are widely distributed in brain regions involved with the modulation of sexual behavior. Here we studied the NADPH-diaphorase (NADPH-d) activity and the number of positive cells in the MePD of adult males and adult females either across the estrous cycle (diestrus, proestrus, estrus, and metaestrus) or following ovariectomy and substitutive therapy (consisting of oil, estradiol alone, the combination of estradiol and progesterone, or progesterone alone). The NADPH-d histochemical technique was followed by a semi-quantitative analysis using optical densitometry. Males showed a higher MePD regional optical density and neuronal optical density than females across the estrous cycle, with the exception of the diestrus phase (P<0.01). No differences were found in these parameters during the ovarian cycle (P>0.05). There were no statistically significant differences among males and cycling females in the number of NADPH-d positive cells (P>0.05). Additionally, no statistically significant difference was found in the regional optical density, in the neuronal optical density, or in the number of NADPH-d positive neurons when comparing the data from ovariectomized females that received vehicle or the three different hormonal replacement therapies (P=0.07, P=0.18, and P=0.95, respectively). Results suggest that NADPH-d activity in the rat MePD is different between sexes but in females it is not affected by changing levels of circulating gonadal hormones in physiological or supraphysiological conditions.