Brains of mice pretreated with saline or 40 micrograms of acetylcholine (ACh) i.c.v. were fractionated according to published procedures. The fractions yielded four peaks of inhibitory activity in the radioreceptor assay. Intraventricular ACh decreased the inhibitory activity of peak I (fractions 10-19), increased that of peak II (fractions 20-24) and peak III (fractions 25-29) and did not change the activity of peak IV in the radiotracer binding assay. Peaks I, III and IV were potent inhibitors of the coaxially stimulated guinea-pig ileum and such inhibitory activity was not destroyed by incubation with trypsin, carboxypeptidase or by naloxone. Intraventricular ACh did not alter the activity of the three peaks on coaxially stimulated ileum bioassay. Peaks II and III both caused a contraction of the nonstimulated guinea-pig ileum and their effect was reduced either by enzymatic treatment (peak II) or by atropine (peak III). No difference was observed between the effects of each peak in saline or ACh-treated mice in this test. All four peaks were active in the writhing test. The results suggest the presence of several opiate-like materials in the brain. The endogenous opioids appear to be a mixture of endorphin-like peptides as well as nonpeptides. The data also indicate the presence of spasmogenic peptides with some opiate properties.