Chronic opiate administration results in the development of tolerance and dependence, but the regulation of mu opioid receptor function during this process is not clearly understood. To localize changes in mu opioid receptor-coupled G-protein activity in various brain regions after chronic morphine treatment, the present study examined mu opioid agonist-stimulated [35S]GTPgammaS binding to brain sections by in vitro autoradiography. Rats were treated for 12 d with increasing doses (10– 320mg . kg-1 . d-1) of morphine. Control rats were injected with either saline or a single acute injection of morphine (20 mg/kg). mu opioid- stimulated [35S]GTPgammaS binding was measured by autoradiography of brain sections in the presence and absence of the mu opioid-selective agonist DAMGO. In rats injected with a single acute dose of morphine, no significant changes were detected in basal or agonist-stimulated [35S]GTPgammaS binding in any region. In sections from chronic morphine- treated rats, however, DAMGO-stimulated [35S]GTPgammaS binding was reduced significantly compared with control rats in the following brain- stem nuclei: dorsal raphe nucleus, locus coeruleus, lateral and medial parabrachial nuclei, and commissural nucleus tractus solitarius. No significant changes were observed in several other brain regions, including the nucleus accumbens, amygdala, thalamus, and substantia nigra. These data indicate that chronic morphine administration results in reductions in mu opioid activation of G-proteins in specific brainstem nuclei involved in physiological homeostasis and autonomic function, which may have implications in the development of opiate tolerance and physical dependence.