Microinjection of opiates into either the periaqueductual gray, locus coeruleus, nucleus raphe magnus, or nucleus reticularis gigantocellularis elicits a profound naloxone-sensitive analgesia. mu-Opioid receptors have been implicated in supraspinal analgesia and studies from our laboratory have demonstrated the importance of the mu 1-receptor subtype. In an effort to examine the receptor subtypes responsible for opioid analgesia in specific brain regions, we examined dose-response relationships and naloxonazine sensitivity of morphine and two enkephalin derivatives in the above 4 brain regions. Both morphine and [D-Ser2,Leu5]enkephalin-Thr6 (DSLET) were effective analgesics in all regions examined. The poor affinity of DSLET for mu 2-receptors and of morphine for delta-receptors, combined with their similar, high affinity for mu 1-receptors, implied a mu 1-mechanism of action. The mu 1-selective antagonist naloxonazine effectively blocked the analgesic responses of both compounds in all regions. [D-Pen2,D-Pen5]enkephalin (DPDPE), a potent delta-ligand which does not interact with mu 1-receptors, did not elicit analgesia in either the periaqueductal gray or locus coeruleus at any dose tested. These results suggest that opiates and opioid peptides produce analgesia in these 4 brain regions through mu 1-receptors. The inactivity of DPDPE argues against a role for delta-receptors and the similar analgesic potencies of morphine and DSLET makes a significant role for mu 2-receptors unlikely.