Several histochemical and physiological studies in the literature suggest that nitric oxide (NO) is involved in the posterior pituitary regulation. This study was set out to demonstrate the distribution of the pituitary projecting nitric oxide containing hypothalamic pathways. The fluorescent retrograde tracer fluorogold (FG) was injected into the pituitary gland in order to reveal the hypothalamic nuclei projecting to the pituitary gland. Nitric oxide synthase (NOS) was subsequently visualized from the same sections by NADPH-diaphorase histochemistry. More than 70% of the FG labelled neurons in the preoptic area, paraventricular, supraoptic, retrochiasmatic, anterior commissural and circular nuclei as well as in many other small neurosecretory centers in the hypothalamus contained also NADPH-diaphorase activity (NADPH-DA). In addition, some of the neurons in the periventricular nucleus as well as occasional neurons in the lateral hypothalamus projecting to the pituitary gland contained NADPH-DA. The present results give the first systematic neuroanatomical description of the hypothalamic NO synthase containing neurons projecting into the pituitary gland. They indicate that the pituitary receives a widely distributed NO innervation, which originates mostly in the magnocellular neurosecretory hypothalamo-pituitary system. In addition, the finding that some presumably non-neurosecretory pituitary-projecting neurons contain NO as well suggests that NO might be a more widely used regulator of the posterior pituitary secretion than previously expected.