Present study aims to clarify the phosphorylation patterns of proteins derived from the rat hypothalamus pretreated with forskolin and phorbol ester in vitro or ovarian steroids in vivo. Proteins were employed to cell free phosphorylation with γ-^(32) P-ATP, analysed by SDS-polyacryl-amide gel electrophoresis and autoradiography. Treatments of forskolin and phorbol ester resulted in intensifying the specific phosphorylation bands of 43 Kd and 130 Kd, respectively, In vivo treatment of ovarian teroids such as estrogen and/or progesterone were highly effective in modifying the phosphorylation patterns of hypothalamic proteins. Furthermore, changes in protein phosphorylation by forskolin, phorbol ester and ovarian steroids were very similar to those with kinase activators such as cAMP, Ca^(2+)-phosphatidylserine and Ca^(2+)-calmodulin. Therefore, it appears that protein phosphorylation is an important step in regulating the trans-synaptic signal transduction in the hypothalamus.