The intracerebroventricular (i.c.v.) injection to mice of antisera directed against different sequences of Gi3 alpha, impaired the antinociception produced by the selective ligands of delta opioid receptors DPDPE and [D-Ala2]-Deltorphin II, when studied 24 h later in the tail-flick test. Likewise, the potency of the mu/delta ligands DADLE, etorphine and beta-endorphin-(1-31) was also reduced. Antinociception due to the mu-agonists morphine and DAMGO was slightly altered by this treatment. The selective delta antagonist ICI 174864 significantly reduced the antinociceptive activity of these opioids to the same extent observed after giving anti-Gi3 alpha antisera. In animals treated with the antisera, ICI 174864 failed to reduce the antinociceptive effect that remained. It is concluded that Gi3 is the type of transducer protein regulated by delta opioid receptors to produce supraspinal antinociception in mice.