In neurons and transformed cell lines, opioid receptors are coupled to various signaling mechanisms involved in Ca2+ mobilization, including inhibition or activation of Ca2+ channels and phospholipase C-beta (PLC-beta), the enzyme responsible for generation of the Ca2+ mobilizing messenger inositol-1,4,5-trisphosphate [Ins(1,4,5)P3]. In the current study, we used selective PLC-beta and G protein antibodies to identify the PLC-beta isozyme activated by opioid receptors in intestinal smooth muscle and the G proteins to which the PLC-beta isozyme and adenylyl cyclase are coupled. [D-Pen2,D-Pen5]Enkephalin, a delta receptor agonist, stimulated Ins(1,4,5)P3 formation, Ca2+ release, and contraction; inhibited forskolin-stimulated cAMP formation in dispersed muscle cells; and stimulated phosphoinositide hydrolysis in plasma membranes; all of the effects were blocked by pertussis toxin. [D-Pen2,D-Pen5]Enkephalin-stimulated Ins(1,4,5)P3 formation, Ca2+ release, and contraction in permeabilized muscle cells and phosphoinositide hydrolysis in plasma membranes were selectively blocked by G beta antibody and PLC-beta 3 antibody; contractions stimulated by [D-Ala2,N-MePhe4,Gly-ol5]enkephalin, a mu receptor agonist, and U-69,593, a kappa receptor agonist, were also blocked by G beta and PLC-beta 3 antibodies. Inhibition of forskolin-stimulated cAMP formation by delta, mu, and kappa receptor agonists was partially blocked by G alpha i2 and G alpha o antibodies and additively blocked by a combination of the antibodies. The delta, mu, and kappa receptor agonists stimulated the binding of guanosine-5'-O-(3-thio)triphosphate to the alpha subunits of Gi2 and G(o) but not to the alpha subunits of other G proteins. We conclude that opioid mu, delta, and kappa receptors are selectively coupled to Gi2 and G(o) in intestinal smooth muscle. The beta gamma subunits of both G proteins activate PLC-beta 3, thereby stimulating Ins(1,4,5)P3-dependent Ca2+ release and smooth muscle contraction, whereas the alpha subunits inhibit adenylyl cyclase activity.