In rats with microdialysis probes in the nucleus accumbens (NAc) or prefrontal cortex (PFC), intraperitoneally (IP) delivered morphine on the 8th day of escalating doses decreased extracellular ACh in the NAc. On day 9, naloxone (5 mg/kg) precipitated withdrawal and increased the release of ACh. When morphine and methylnaloxonium were given locally into the NAc by reverse dialysis, the opiate again decreased extracellular ACh, and the opiate antagonist increased it. These effects were proportional to the dose of local infusions. Local morphine had the same ACh-lowering effect in morphine-dependent and nondependent rats, whereas local methylnaloxonium increased extracellular ACh significantly more in morphine-dependent animals. Systemic and local effects on ACh systems in the PFC were more complicated and showed some relation to locomotor activity. The results suggest that intrinsic ACh neurons in the NAc have a special relationship to opiate reinforcement such that extracellular ACh is low in response to morphine and high during withdrawal. Thus, low ACh may correlate with opiate reward, and high ACh with aversion.