This laboratory previously reported that the mu-selective opiate receptor agonist, sufentanil, produces a naloxone-reversible, concentration-dependent facilitation or inhibition of the stimulated formation of cAMP in the myenteric plexus. Chronic in vivo exposure to morphine results not only in the loss of inhibitory opioid responsiveness but in the reversal of inhibition to enhancement. The present study demonstrates, in tolerant/dependent as well as opiate naive tissue, that the state of phosphorylation is a critical determinant of the balance between positive and negative opioid modulation of stimulated cAMP formation. In vitro treatment of chronic morphine-treated preparations with inhibitors of protein kinases, abolishes the previously observed reversal of opioid inhibition to enhancement and restores sufentanil inhibitory responsiveness. The established kinase-type selectivity profile of the inhibitors employed suggests the involvement of protein kinase C (PKC) in the tolerant-associated reversal from opioid inhibition to enhancement of cAMP formation. Conversely, treatment of opiate naive tissue with the protein phosphatase inhibitor okadaic acid or a phorbol ester activator of protein kinase C, phorbol 12-myristate 13-acetate (PMA), not only attenuates sufentanil inhibition of evoked cAMP formation but reverses it to a facilitation (as occurs following chronic in vivo morphine exposure). This effect of PMA is abolished by the PKC-selective inhibitor chelerythrine. Moreover, the longitudinal muscle myenteric plexus content of PKC alpha and PKC beta is substantially elevated following chronic morphine treatment. These results underscore the relevance of opioid bimodality to the manifestation of tolerance/dependence and suggest that augmented phosphorylation (mediated at least in part via PKC) is a critical determinant of some of the sequelae of chronic morphine exposure.