Evaluation of the effects of tolerance to morphine in vivo on free intracellular calcium levels [(Ca++)i] in whole brain synaptosomes from rats and synaptosomes from brain regions and spinal cords of both rats and mice indicated that in whole brain and spinal cord synaptosomes, [Ca++]i levels are significantly higher in morphine-tolerant mice. Although levels do not differ between nontolerant and tolerant preparations from the rat spinal cord, [Ca++]i brain levels are significantly higher in morphine-tolerant rats. Evaluation of three brain regions from both nontolerant and tolerant rats and two brain regions from mice indicated no significant differences in basal [Ca++]i, with the exception that lower basal levels of calcium were observed in the midbrain of the tolerant rat. In vivo, mice tolerant to morphine (SC) were cross tolerant to DAMGO (IT and ICV). Cross tolerance between morphine and DAMGO was observed in vitro. No cross-tolerance was observed between morphine (SC) and the delta-agonist, DPDPE (IT or ICV), or the kappa-agonist, U50,488H (U50, IT or ICV) in vivo. Similarly, no cross tolerance was observed between morphine (SC) and DPDPE or U50 in vitro. These data indicate that tolerance in vivo induced a change in synaptosomal calcium modulation such that tolerance could be observed in vitro. Thus, a membrane component may be altered by the development of tolerance to morphine leading to an alteration in [Ca++]i in both the brain and spinal cord.