Vasoactive intestinal polypeptide (VIP) has a markedly heterogeneous distribution in the rat bed nucleus of the stria terminalis. The dorsal bed nucleus contains the highest concentration of VIP in the rat brain, with the exception of the suprachiasmatic nucleus, 4-fold higher than the VIP concentration in the frontal cortex. These biochemical findings agree well with the immunohistochemical analysis of this area. The bed nucleus is also a heterogeneous nucleus with respect to the afferent VIP pathways which innervate it. A combination of immunohistochemical and biochemical techniques was used to examine VIP innervation of the bed nucleus after knife cuts designed to interrupt ascending brainstem, stria terminalis and ventral amygdalofugal inputs to the bed nucleus. The results obtained suggest that (1) ascending pathways arising in the mesencephalon at the level of the dorsal raphe nucleus send VIP fibers to the dorsal but not the ventral bed nucleus, (2) afferent VIP fibers which travel to the bed nucleus via the stria terminalis contribute a diffuse VIP innervation to both the dorsal and ventral bed nucleus and (3) a newly described ventral amygdalofugal VIP pathway to the bed nucleus contributes a major input to the dorsal, but not to the ventral bed nucleus. These three pathways probably account for the entire extrinsic VIP input to the bed nucleus. The finding that the bed nucleus is heterogeneous both with respect to VIP content and afferent VIP inputs serves to clarify previous, apparently discrepant, reports that both the stria terminalis and ascending pathways constitute the major VIP input to the bed nucleus of the stria terminalis.