Male rat copulatory ability decreases dramatically following castration. This may be due in part to the impairment of medial preoptic area (MPOA) dopamine (DA) release. Previous studies showed that extracellular DA levels in the MPOA of castrates were lower than in intact males, both during basal conditions and in the presence of a receptive female. However, tissue levels of DA in the MPOA were higher in castrates than in intact males, suggesting that DA synthesis may be normal or increased in castrates, but that release may be compromised. The current study found that neither long term (2 months) nor short term (2 weeks) castration had any effect on the number of neurons in the DA A 14 area that were immunoreactive (ir) for tyrosine hydroxylase (TH), the rate limiting enzyme for DA synthesis. Therefore, castration may not affect DA synthesis in the MPOA. Tissue levels of neurotransmitter reflect release, as well as synthesis. We previously reported that nitric oxide (NO) may increase DA release in the MPOA. The present study tested whether castration affected the number of NO producing cells in the MPOA. Long term, but not short term, castration significantly decreased the number of NADPH-d (nicotinamide adenine dinucleotide phosphate diaphorase) positive neurons and brain nitric oxide synthase immunoreactive (bNOS-ir) neurons in the medial preoptic nucleus (MPN). This suggests that in gonadally intact animals testosterone may activate NOS, which increases the production of NO. Long or short term castration had no effect on the numbers of bNOS-ir neurons in the paraventricular nucleus (PVN) or medial amygdala. However, short term castration decreased bNOS-ir neurons in the bed nucleus of stria terminalis (BNST). Thus, one means by which testosterone promotes male sexual behavior may be by increasing production of NO in the MPOA, which increases local DA release.