In the current study, we investigated the neurochemical basis for the previously reported predominance of stimulatory mu-opioid signaling in guinea pig longitudinal muscle/myenteric plexus (LMMP) preparations after chronic in vivo morphine exposure. As expected, recombinant Gsalpha (rGsalpha) dose-dependently stimulated adenylyl cyclase (AC) activity in LMMP membranes obtained from opioid naive as well as tolerant LMMP tissue. However, the magnitude of the increase was significantly greater in the latter than in the former. The Gbetagamma blocking peptide QEHA (50 microM) essentially abolished stimulation by rGsalpha in LMMP membranes obtained from both opioid naive and tolerant animals. Interestingly, after partial blockade by lower QEHA concentrations, the incremental AC stimulation by rGsalpha in tolerant LMMP membranes was no longer observed, indicating augmented Gbetagamma stimulatory responsiveness. Concomitant changes in the content of AC isoform protein are consistent with these biochemical observations. After chronic systemic morphine, AC protein is augmented significantly (56%). This increment is most likely to be composed of AC isoforms that are stimulated by Gbetagamma. This is the first demonstration in a complex mammalian tissue that persistent activation of opioid receptors results in augmented Gbetagamma/Gsalpha AC stimulatory interactiveness. The relevance of such changes to the manifestation of opioid tolerance is discussed.