Although the mu selective agonist [D-Ala2-MePhe4-Gly-ol5]enkephalin (DAMGO) and the delta selective agonist [D-Pen2,D-Pen5]enkephalin (DPDPE) are both antinociceptive when administered directly into the spinal cord of mice, 50% of antinociceptive dose (AD50) of DAMGO is about 2 orders of magnitude lower than the AD50 of DPDPE. In contrast, the two ligands show similar affinities for their respective receptors in in vitro binding assays. One possible explanation for this discrepancy is that DPDPE antinociception in the spinal cord is mediated through not delta but mu receptors, for which it has an several hundred-fold lower affinity than DAMGO. In support of this, we found that DPDPE-mediated antinociception was blocked by the mu selective antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP). The pA2 value of CTAP for DPDPE was virtually identical with that for DAMGO. However, because its action also was blocked by naltrindole, an antagonist selective for delta receptors, the latter must also play a role in antinociception. When DAMGO and DPDPE were administered i.t. together at ratios ranging from 1:200 to 1:500, the AD50 of DAMGO was lowered as much as 10-fold relative to its AD50 when given alone. Thus DPDPE had a potentiating effect on DAMGO, although the reverse was not observed. This potentiation was lost in animals made tolerant to systemic morphine. The loss of potentiation seemed to be caused by changes in the delta receptors, because a) the AD50 of DAMGO (i.t.) given alone to tolerant animals was virtually the same as for naive animals, whereas the AD50 of DPDPE given alone increased by 4-fold; and b) the AD50 of DPDPE given alone in the tolerant animal was increased only slightly by naltrindole, whereas CTAP was still a very potent antagonist. We conclude that DPDPE, a selective delta agonist, mediates antinociception in the spinal cord through mu receptors, consistent with results of recent studies of "knock-out" mice lacking mu receptors. At the same time, however, the delta agonist acting through delta receptors can potentiate the mu receptor-mediated antinociceptive action of either mu or delta agonists. This potentiating effect, like the synergistic effect observed between mu receptors at spinal and supraspinal sites, is lost during tolerance.