The subcellular localization of in vivo synthesized cholecystokinin (CCK) in different parts of the rat brain was studied after intracisternal pulse injections of [35S]methionine. The rats were decapitated 1 h after the injection, and the brain was divided into cortex, hippocampus and remainder. Subcellular fractions were obtained according to Whittaker's method. De novo synthesized CCK in the crude mitochondrial-synaptosomal fraction, P2, and in the purified synaptosomal fraction was demonstrated by affinity chromatography, using antibodies specific for the COOH-terminal sequence of CCK. By subsequent gel chromatography two molecular forms of labelled CCK occurred, with elution constants, Kav, of 1.1 (corresponding to the COOH-terminal octapeptide) and of 1.40 (a component which may correspond to the COOH-terminal tetrapeptide amide, CCK-4). The findings support the idea that the small molecular forms are the transmitter forms of CCK.