When coadministered spinally, morphine and clonidine interact synergistically to produce antinociception. The mechanism for the synergism is unknown, but may depend on intracellular messenger systems. Agents that alter the activities of protein kinases alter antinociception produced by opioids, but their effects on clonidine-induced antinociception or the morphine/clonidine interaction are not known. In these studies, mice were pretreated intrathecally with inhibitors or activators of protein kinase C and cyclic AMP-dependent protein kinase (protein kinase A). Antinociceptive responses to intrathecally administered morphine, clonidine and morphine/clonidine combinations were then measured in the radiant heat tail flick test. Inhibition of protein kinase C activity with chelerythrine or calphostin C changed the morphine/clonidine interaction from synergistic to additive. Inhibition of protein kinase A activity with H-89 did not alter the morphine/clonidine interaction, it remained synergistic. Stimulation of protein kinase C activity with phorbol 12,13-dibutyrate attenuated morphine antinociception, but did not alter the synergistic interaction. Increasing spinal cyclic AMP concentrations with either forskolin or rolipram attenuated the antinociception produced by separately administered morphine and clonidine, but had no effect on the morphine/clonidine interaction. These results suggest that protein kinase C activity may regulate the interaction between spinal opioid and alpha-2 receptors, stimulated by morphine and clonidine.