This laboratory has demonstrated that the longitudinal muscle/myenteric plexus (LMMP) preparation manifests pleiotropic responses to opioid agonists. For example, the mu-selective opiate receptor agonist sufentanil can produce a naloxone-reversible increase or decrease in the electrically stimulated formation of cyclic AMP, depending on its concentration. The present study demonstrates that the sufentanil facilitation and inhibition of stimulated cyclic AMP formation are mediated via Gs- and Gi-like G proteins, respectively. Inactivation of Gi (via pertussis toxin) not only abolishes sufentanil inhibition of cyclic AMP formation but also unmasks a facilitory effect. The latter response is eliminated following treatment with cholera toxin. In tolerant/dependent LMMP tissue, previously inhibitory concentrations of sufentanil produce a facilitation of cyclic AMP formation. However, this unmasked facilitory effect is resistant to cholera toxin. Thus, although inactivation of the inhibitory signal transduction pathway (via pertussis toxin) is sufficient to unmask excitatory sufentanil effects in opiate naive preparations, this mechanism cannot explain the reversal of sufentanil inhibition to facilitation that is observed in tolerant/dependent tissue. Instead, the chronic morphine-induced emergence of a mu-opiate receptor-coupled facilitory pathway that is either not expressed or not fully manifest in opiate naive LMMP tissue is suggested.