Delta-selective agonists have been developed to produce potent analgesic compounds with limited side-effects. DPDPE and deltorphin II are considered prototypes, but their delta-selectivity in vivo and the true ability of delta receptors to produce analgesia remain to be demonstrated. Here we have performed a parallel analysis of mu, delta and combinatorial opioid receptor knockout mice, in which we found no obvious alteration of G-protein coupling for remaining opioid receptors. We compared behavioural responses in two models of acute thermal pain following DPDPE and deltorphin II administration by intracerebroventricular route. In the tail-immersion test, both compounds were fully analgesic in delta knockout mice and totally inactive in mu knockout mice. In the hotplate test, the two compounds again produced full analgesia in delta knockout mice. In mu knockout mice, there was significant, although much lower, analgesia. Furthermore, DPDPE analgesia in the delta knockout mice was fully reversed by the mu selective antagonist CTOP in both tests. Together, this suggests that mu rather than delta receptors are recruited by the two agonists for the tail withdrawal and the hotplate responses. Finally, deltorphin II slightly prolonged jump latencies in double mu/kappa knockout mice (delta receptors only) and this response was abolished in the triple knockout mice, demonstrating that the activation of delta receptors alone can produce weak but significant mu-independent thermal antinociception.