In previous studies from our laboratory, chronic noncontingent morphine administration decreased μ opioid receptor-activated G-proteins in specific brainstem nuclei. In the present study, μ opioid receptor binding and receptor-activated G-proteins were examined after chronic heroin self-administration. Rats were trained to self-administer intravenous heroin for up to 39 d, achieving heroin intake up to 366 mg · kg⁻¹ · d⁻¹. μ opioid-stimulated [³⁵S]GTPγS and [³H]naloxone autoradiography were performed in adjacent brain sections. Agonist-stimulated [³⁵S]GTPγS autoradiography also examined other G-protein-coupled receptors, including δ opioid, ORL-1, GABA_B, adenosine A₁, cannabinoid, and 5-HT_1A. In brains from heroin self-administering rats, decreased μ opioid-stimulated [³⁵S]GTPγS binding was observed in periaqueductal gray, locus coeruleus, lateral parabrachial nucleus, and commissural nucleus tractus solitarius, as previously observed in chronic morphine-treated animals. In addition, decreased μ opioid-stimulated [³⁵S]GTPγS binding was found in thalamus and amygdala after heroin self-administration. Despite this decrease in μ-activated G-proteins, [³H]naloxone binding demonstrated increased μ opioid receptor binding in several brain regions after heroin self-administration, and there was a significant decrease in μ receptor G-protein efficiency as expressed as a ratio between agonist-activated G-proteins and μ receptor binding. No effects on agonist-stimulated [³⁵S]GTPγS binding were found for any other receptor examined. The effect of chronic heroin self-administration to decrease μ-stimulated [³⁵S]GTPγS binding varied between regions and was highest in brainstem and lowest in the cortex and striatum. These results not only provide potential neuronal mechanisms that may contribute to opioid tolerance and dependence, but also may explain why various chronic effects of opioids develop to different degrees.