The effects of opiate agonists on acetylcholine release from hippocampal, striatal and cerebral cortical slices were tested; tissue from rat was compared to that from guinea-pig. The results show that opiate receptors in each of these areas can alter the evoked release of acetylcholine from nerve terminals; however, there are species and tissue differences with respect to the apparent subtype of opiate receptor effective. In the hippocampus and striatum of the two species studied, opiates caused a dose-dependent decrease in evoked acetylcholine release from tissue slices but in the guinea-pig kappa-selective agonists were effective, and mu or delta agonists were not, whereas in the rat, mu-, but not delta- or kappa-selective drugs were effective. Opiates also altered acetylcholine release from the frontal, parietal and occipital cortex of both of these species. In all three regions of the guinea-pig cortex, kappa and delta agonists were active and in the parietal cortex mu agonists were also active; rat cortical slices showed similar results except that delta agonists were not effective. The inhibitory effects of the opiate agonists were effectively antagonized by the non-selective opiate antagonist naloxone and by the calcium channel agonists, BAY K 8644 or YC-170. In addition, the effects of the opiate drugs tested in this study on acetylcholine release were confined to evoked release, that is, spontaneous acetylcholine release was not affected. The results suggest that in guinea-pig and rat brain, opiate receptors regulate acetylcholine release, and that, although the subtypes of opiate receptors involved in this effect are different in the two species and in different tissues from the same species, the effect results from a common mechanism that involves alterations of calcium influx into the nerve terminals during depolarization.