Cholecystokinin-like immunoreactivity (CCK-LI) was demonstrated by radioimmunoassay in aqueous (n = 3) and acid (n = 10) extracts of cortex (42 +/- 9 pmol/g; 4.0 +/- 1.8 pmol/g), thalamus (4.1 +/- 1.1 pmol/g; 1.0 +/- 0.2 pmol/g), and hypothalamus (58 +/- 14 pmol/g; 6.3 +/- 0.7 pmol/g). Sephadex chromatography revealed that more than 95% of the immunoreactivity in acid extracts coeluted with CCK33 standard. In aqueous extracts more than 80% of immunoreactivity coeluted with CCK8 standard. Both the CCK33- and CCK8-like materials induced contraction of guinea pig gallbladder in vitro. L-Tryptophan (200 mg/kg) and high-dose morphine (20 mg/kg) decreased CCK33-LI concentrations in hypothalamus and thalamus. Low-dose morphine (5 mg/kg) decreased CCK33-LI in hypothalamus. We conclude that 1) CCK-LI is present in cortex, thalamus, and hypothalamus of the rat brain, 2) CCK-LI exists in two predominant molecular forms coeluting with CCK33 and CCK8, 3) both molecular forms are biologically active, and 4) concentrations of rat brain CCK33-LI are modulated by serotonergic and opiate mechanisms.