Opioid analgesic tolerance is associated with a disruption in Ca++ homeostasis. Drugs reducing Ca++ influx can prevent and reverse tolerance. The hypothesis was tested that both Ca++ influx and mobilization from intracellular pools maintains the expression of morphine tolerance. Ca++ modulating drugs were injected ICV at doses not affecting morphine's potency in placebo pellet-implanted mice, in order to determine whether tolerance would be reversed in morphine pellet-implanted mice. The Ca++ chelator EGTA significantly reversed tolerance. The Ca++ channel antagonists nifedipine and omega-conotoxin GVIA also reversed tolerance. The role of intracellular Ca++ was investigated using the membrane permeable intracellular Ca++ chelator EGTA-AM. EGTA-AM reversed tolerance at lower morphine doses, but not at higher morphine doses. Thus, mobilization of intracellular Ca++ contributes to the expression of tolerance. Finally, 1,4-dihydropyridine-sensitive Ca++ channels are known to stimulate Ca++-induced Ca++ release (CICR) from Ca++/caffeine-sensitive microsomal pools possessing ryanodine receptors. We examined whether blocking Ca++ mobilization from these pools with ryanodine would reverse morphine tolerance. Ryanodine's effects were similar to EGTA-AM. Tolerance was reversed at lower morphine doses, but not at higher doses. Thus, morphine tolerance appears to be associated with increases in Ca++ influx and mobilization from Ca++/caffeine-sensitive pools.