The effect of electrical stimulation of the prefrontal cortex (PFC) on acetylcholine (ACh) release in the dorsal striatum was investigated using on line in vivo microdialysis. ACh output was sampled before, during and after 20-min periods of 50 and 100 microA stimulation in awake, unrestrained rats. Both currents increased extracellular ACh by approximately 30% above baseline. ACh concentrations reached their maximum values during the last 10 min of the stimulation period and returned to baseline within 20 min. These results provide direct functional evidence for cortical modulation of cholinergic interneurons in the striatum.