The concurrent use of cocaine and heroin, often referred to as speedball, is a powerful reinforcer that has been reported in humans to sometimes result in heightened euphoria compared with either drug alone. Data from animal research indicate that the reinforcing efficacy of low doses of cocaine is potentiated by the addition of small amounts of heroin and that this potentiation is accompanied by synergistic increases in nucleus accumbens (NAc) extracellular fluid levels of dopamine. Although micro- and/or delta-opioid receptors may underlie this potentiation, the opioid receptor subtype or the loci responsible for this enhancement is not known. This experiment used intracranial administration of a selective micro-opioid receptor alkylating agent (beta-funaltrexamine (beta-FNA)) to assess the role of mu-opioid receptors in the NAc, ventral pallidum (VP), and ventral tegmental area (VTA) on the ability of heroin to alter cocaine self-administration. Rats were trained to self-administer cocaine, heroin, or their combination and were administered either vehicle or beta-FNA into one of each brain region and the effects upon drug intake assessed. beta-FNA administered into the VP or VTA shifted the dose-effect curve for the cocaine/heroin combination towards that maintained by cocaine alone. beta-FNA had no effect on self-administration of the combination of cocaine and heroin when injected into the NAc. These data suggest that heroin may attenuate feedback inhibition from the NAc to the VP and VTA when co-self-administered with cocaine, resulting in a positive modulation of the effects of cocaine.