Once daily s.c. administration of 5 mg/kg morphine, a mu-opioid agonist, or U50488H (U50), a kappa1-opioid agonist, for 5 days in male CD-1 mice results in a 2–3-fold shift to the right of the respective analgesic (tail flick) dose-response curves, indicating the development of tolerance. Concurrent s.c. administration of the competitive NMDA receptor antagonist, LY274614 (LY), at 24 mg/kg/24 h infusion (osmotic pump) or 6 mg/kg i.p. once daily attenuates the development of morphine tolerance, when the response to saline plus morphine is compared on day 5 with LY plus morphine. Using this paradigm, once daily administration of either the non-competitive NMDA antagonist, MK-801, at 0.3 mg/kg i.p. or the nitric oxide synthase inhibitor, NG-nitro-L-arginine (NorArg), at 1 mg/kg i.p. twice daily attenuated the development of morphine tolerance. None of these drugs modify the tail-flick response or alter the ED50 for morphine. In contrast, co-administration of LY, MK-801 or NorArg, as above, failed to attenuate the development of tolerance to U50 or to the kappa3-opioid agonist, naloxone benzoylhydrazone (NalBzoH). These results suggest that mu-opioid tolerance but not kappa1- or kappa3-opioid tolerance involves the mediation of NMDA receptors and the nitric oxide system.