1. Opioid tolerance involves an alteration in the activity of intracellular kinases such as cyclic AMP-dependent protein kinase (PKA). Drugs that inhibit PKA reverse morphine antinociceptive tolerance. The hypothesis was tested that phospholipid pathways are also altered in morphine tolerance. Inhibitors of the phosphatidylinositol and phosphatidylcholine pathways were injected i.c.v. in an attempt to acutely reverse morphine antinociceptive tolerance. 2. Seventy-two hours after implantation of placebo or 75 mg morphine pellets, mice injected i.c.v. with inhibitor drug were challenged with morphine s.c. for generation of dose-response curves in the tail-flick test. Placebo pellet-implanted mice received doses of inhibitor drug having no effect on morphine's potency, in order to test for tolerance reversal in morphine pellet-implanted mice. Injection of the phosphatidylinositol-specific phospholipase C inhibitor ET-18-OCH3 significantly reversed tolerance, indicating a potential role for inositol 1,4,5-trisphosphate (IP3) and protein kinase C (PKC) in tolerance. Alternatively, phosphatidylcholine-specific phospholipase C increases the production of diacylglycerol and activation of PKC, without concomitant production of IP3. D609, an inhibitor of phosphatidylserine-specific phospholipase C, also reversed tolerance. Heparin is an IP3 receptor antagonist. Injection of low molecular weight heparin also reversed tolerance. PKC was also examined with three structurally dissimilar inhibitors. Bisindolylmaleimide I, Go-7874, and sangivamycin significantly reversed tolerance. 3. Chronic opioid exposure leads to changes in phospholipid metabolism that have a direct role in maintaining a state of tolerance. Evidence is accumulating that opioid tolerance disrupts the homeostatic balance of several important signal transduction pathways.