Physiological evidence indicates that the supraoptic nucleus may be an important integrating region for information relating to body fluid homeostasis. It is known that the supraoptic nucleus receives neural influences from brain receptive zones for plasma osmolality and angiotensin II, as well as from relay centers for blood pressure and blood volume. It is also known that these influences interact to modulate vasopressin release from the supraoptic nucleus. Therefore, a detailed investigation of the neurochemical afferents to the supraoptic nucleus from regions of the lamina terminalis and the brainstem was undertaken. Injection of a fluorescent retrograde tracer, doxorubicin, into the supraoptic nucleus was combined with histochemistry of angiotensin II and catecholamines. Following supraoptic nucleus injection, retrograde label was found in forebrain neurons of the subfornical organ, median preoptic nucleus, and organum vasculosum of the lamina terminals. Some labeled cells in the subfornical organ and organum vasculosum of the lamina terminalis were also found to contain angiotensin II immunoreactivity. In the brainstem, retrograde label was found in neurons of the A1, A2 and A6 cell groups. Many of these cells were also found to contain catecholamine fluorescence or tyrosine hydroxylase immunoreactivity. Corroboration of the A2 projection was obtained by lesions of this nucleus, which reduced catecholamine fluorescence in the supraoptic nucleus. These findings provide an anatomical basis for the functional observations that the supraoptic nucleus plays a key integrative role in the maintenance of body fluid homeostasis.