Morphine antinociceptive tolerance in the tail-flick test is completely reversed by inhibitors of protein kinase C (PKC) or cAMP-dependent protein kinase (PKA). The effects of these inhibitors on tolerance to supraspinally mediated antinociception, such as the hot-plate test was unknown, as well as their effects in tests of mechanical nociception. The PKC inhibitors bisinolylmaleimide I ((2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) and Gö-7874 [2[1[(3-Dimethylaminopropyl)-5-methozyindol-3-yl]-3-(1H-indol-3-yl) hydrochloride] completely reversed the tolerance to morphine in both the hot-plate and tail-pinch tests. Similarly, the PKA inhibitor KT-5720 (8R, 9S, 11S)-(-)-9-hydroxy-9-hexoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinden-1-one also reversed tolerance in both tests. The role of PKC and PKA in mediating tolerance to morphine-induced hypothermia was also investigated. Bisinolylmaleimide I, Gö-7874 and KT-5720 only partly reversed the 32-fold level of tolerance induced by the morphine pellets. However, co-administration of bisinolylmaleimide I with KT-5720 or Gö-7874 with KT-5720 completely reversed the tolerance. This demonstrates that tolerance in a non-behavioral system involves the actions of PKC and PKA.