Circadian changes in cortical acetylcholine (ACh) release were examined in freely moving rats using long-term in vivo microdialysis and high performance liquid chromatography with electrochemical detection. A microdialysis probe was implanted into the frontal cortex and a dialysate was collected every 30 min for two or three days under a 12 h light/dark (LD 12:12) cycle and for the succeeding 2 or 3 days under constant dark (DD) conditions. When animals were exposed to the LD cycle, dialysate concentrations of ACh showed a clear circadian rhythmicity with the highest peak in the dark phase and the lowest peak in the light phase. The mean cortical ACh release during the dark phase under the LD cycle was approximately 33% higher than that during the light phase. Under DD conditions, circadian changes in ACh release persisted with similar amplitudes as observed in the LD condition. These results suggest that cortical ACh release is under the control of a circadian oscillator.