Methadone is clinically effective as substitution therapy in patients with opioid dependence. The diversity of methadone and morphine in their intracellular activity is postulated. We compared the effects of repeated daily treatment of Sprague-Dawley rats with morphine (10 mg/kg) and methadone (1 mg/kg) on the expression of the Gα(i1-i3) mRNAs in several rat brain areas using RT-qPCR. We found that both opioid receptor agonists decreased Gα(i3) mRNA in only the nucleus accumbens. Although there was no difference in the influence of morphine and methadone on Gα(i), our results indicate that among Gα(i) subunits, the Gα(i3) is specifically involved in the mechanism of action of both drugs.