Three forms of protein kinase-C were resolved from rat hypothalamus: Types I, II, and III, which correspond to the brain subspecies γ, β, and α, respectively. The rat pituitary contains the type II and type III enzymes but not type I. The hypothalamic type II enzyme is a mixture of βI (24%) and βII (76%), whereas the pituitary type II enzyme contains most likely only the βII enzyme. The hypothalamic type I enzyme is relatively resistant to activation by the tumor-promoting phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) or diacylglycerol (DG). Both type II and type III enzymes of hypothalamus and pituitary were responsive to TPA or DG stimulation, with the hypothalamic subspecies being less responsive. Binding of [3H]phorbol 12, 13-dibutyrate revealed different binding properties among the various subspecies, with the pituitary enzymes displaying higher affinities than the respective hypothalamic counterparts. The results demonstrate heterogeneity in protein kinase-subspecies expression and responsiveness to TPA and DG and suggest specific roles for the subspecies in the hypothalamo- pituitary axis.