Histamine has remained a putative neurotransmitter for many years, partially because some of the criteria necessary to define it as a central nervous system neurotransmitter have not been established. The demonstration of in vitro release and the quantification of turnover as an indirect measure of release have been complicated by the histological evidence for multiple histamine pools in the central nervous system. In brain, there are multiple cell types which probably contain histamine. These cells include mast cells, neurolipomastocytoid cells, microvascular endothelial cells, and a histaminergic neuronal system which has been visualized using immunocytochemical methods. Using in situ brain microdialysis and a sensitive and specific radioenzymatic assay for histamine, we have identified histamine in the extracellular space of the rat hypothalamus and corpus striatum in vivo. Following neuronal selective stimuli, significant increases in extracellular histamine levels only were observed in the posterior hypothalamus, where dense histaminergic neuronal terminals have been described. However, after manipulations targeted towards histamine-containing mast cells, such increases were seen in both the posterior hypothalamus and corpus striatum. In summary, this study demonstrates that endogenous histamine can be released from the posterior hypothalamus by stimuli targeted towards histamine neurons and that histamine may also be released by non-neuronal mast cell elements.