Abstract Acetylcholine (ACh) turnover rates were measured in fourteen brain regions of rats intravenously self-administering morphine and in yoked-morphine and yoked-vehicle infused littermates to identify cholinergic neuronal pathways potentially involved in opiate reinforcement processes. Rats receiving chronic passive administration of morphine had increased ACh turnover rates in the frontal cortex and diagonal band and decreased rates in the medial septum. The significant changes in animals self-administering the drug were prominent in limbic regions with increases in the frontal cortex and decreases in the pyriform cortex, nucleus accumbens, amygdala and ventral tegmental area. Some components of opiate reinforcement may be mediated by increases in the activity of cholinergic ventral pallidal and diagonal band fibers innervating the frontal cortex and by decreases in activity of cholinergic fibers innervating the ventral tegmental area. These data and turnover rates for dopamine, norepinephrine, serotonin, aspartate, glutamate and gamma-aminobutyric acid previously determined in similarly treated animals are consistent with two neuronal circuits that may be involved in opiate seeking behaviors and opiate reinforcement processes.