Neuropeptide Y (NPY) coexists with vasopressin or oxytocin in magnocellular neurons of the hypothalamo-neurohypophysial tract. Using quantitative in situ hybridization histochemistry and immunohistochemistry, we have studied the effects of adrenalectomy and chronic osmotic stimulation, either alone or in combination, on NPY mRNA expression and NPY immunoreactivity in magnocellular neurons of the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, and arcuate nucleus (Arc). Adrenalectomy and chronic osmotic stimulation each increased NPY mRNA levels in magnocellular neurons of the PVN and SON, while the combination of both treatments had an additive effect. In the Arc, only the combination of adrenalectomy and chronic osmotic stimulation increased NPY mRNA levels. Chronic osmotic stimulation also resulted in a marked increase of NPY-immunoreactive magnocellular perikarya in the PVN and SON. In contrast, adrenalectomy had only minor effects on the number of NPY-immunoreactive magnocellular PVN/SON perikarya. Neither chronic osmotic stimulation nor adrenalectomy affected the number of NPY-immunoreactive Arc perikarya. However, adrenalectomy decreased the number of NPY-immunoreactive nerve terminals in the external zone of the median eminence, while chronic osmotic stimulation increased the number of immunoreactive nerve fibers in the internal zone of the median eminence. The present study provides evidence that adrenalectomy and chronic osmotic stimulation can separately influence NPY gene transcription in magnocellular hypothalamo-neurohypophysial neurons, while only the combined effect of adrenalectomy and chronic osmotic stimulation increases NPY mRNA expression in neurons of the Arc.