The tridecapeptide, neurotensin (NT), is heterogenously distributed in the mammalian central nervous system and exhibits many neurotransmitter-like characteristics. However, the molecular mechanisms of NT signal transduction remain obscure. In this report, we demonstrate NT-induced stimulation of specific protein substrate phosphorylation in the rat caudate nucleus. Rat caudate nucleus was dissected, a P2 fraction prepared and proteins phosphorylated in vitro with [32P]ATP for 1 min. Phosphorylated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiograms prepared. NT preincubation in the absence of calcium resulted in markedly increased phosphorylation in vitro of proteins with apparent molecular weights of 80,000 and 50,000. These effects were not observed if calcium was present during the NT preincubation period. Both calcium and cAMP enhanced phosphorylation of the 80 kDa protein, but phosphorylation of the 50 kDa protein was responsive only to calcium.