It has recently been shown that the activation of mu-opioid receptors inhibits several components of calcium channel current in rat DRG sensory neurons. mu-Opioid receptors, acting through the pertussis toxin (PTX)-sensitive substrate Gi, also reduce the activity of neuronal adenylate cyclase, but the relationship of this effect to changes in calcium channel activity has yet to be determined. Using whole-cell recordings from acutely isolated rat DRG neurons, we examined the ability of the mu-opioid-selective agonist Tyr-Pro-NMe-Phe- D-Pro-NH2 (PLO17) to reduce calcium current after treatment with PTX and in the presence of the nonhydrolyzable GTP analog guanosine 5′-[- thio]triphosphate (GTP gamma S), to assess the role of G-proteins in the coupling of mu-opioid receptors to calcium channels. Inhibition of current by PLO17 was mimicked or rendered irreversible by intracellular administration of GTP gamma S, an activator of G-proteins, and was blocked by pretreatment of neurons with PTX. In contrast, when the catalytic subunit of cAMP-dependent protein kinase was included in the recording pipette, calcium currents increased in magnitude throughout the recording without attenuation of responses to PLO17. Thus, the mu- opioid-induced inhibition of calcium current occurs through activation of a Gi- or G(o)-type G-protein, but independent of changes in adenylate cyclase activity. As a first step in identifying this G- protein, we compared the ability of several antisera directed against specific regions of Gi and G(o)alpha subunits to block the inhibition in current by PLO17. Intracellular dialysis with an antiserum specific for G(o) (GC/2) attenuated calcium current inhibition by PLO17 in five of six neurons by an average of 75%. In contrast, there was no attenuation in the response to PLO17 when neurons were dialyzed with an anti-Gi1 alpha/Gi2 alpha antiserum (AS/7) or antibodies specific for alpha subunits of Gi proteins (Gi1/Gi2 or Gi3) in an identical manner. These results suggest that in rat DRG neurons mu-opioid receptors couple to calcium channels via the PTX-sensitive G(o) subclass of GTP- binding proteins.