Chronic activation of opioid receptors in cultured mammalian cells is known to induce adenylyl cyclase (AC) supersensitization via the pertussis toxin-sensitive G(i/o) proteins. To examine the role of G(i1) and G(i3) in opioid-induced AC supersensitization, pertussis toxin-resistant mutants of Galpha(i1) and Galpha(i3) (Galpha(i1)CG and Galpha(i3)CG) were stably co-expressed with different opioid receptors (mu, delta or kappa) in human embryonic kidney (HEK 293) cells. Although the opioid receptors were capable of inhibiting AC via Galpha(i1)CG and Galpha(i3)CG in pertussis toxin-treated cells, AC supersensitization induced by chronic opioid treatment remained sensitive to pertussis toxin. Our results demonstrated that despite their ability to interact with opioid receptors, the pertussis toxin-sensitive G(i1) and G(i3) proteins on their own are incapable of supporting opioid-induced AC supersensitization.