The possible relationship between behavioral arousal and acetylcholine release in the striatum, hippocampus and frontal cortex was investigated in rats. In vivo microdialysate concentrations of acetylcholine and choline from these brain structures, and photocell beam interruptions (as a measure of behavioral arousal), were measured simultaneously under three conditions: after injections of 1) vehicle or 2) scopolamine (0.4 mg/kg), and 3) before and after the beginning of the rats' night cycle. Dialysate concentrations of ACh in all 3 brain structures and locomotor activity were increased after scopolamine and the onset of the lights out condition. Vehicle injections transiently increased ACh in the hippocampus and cortex and caused short-lasting increases in locomotor activity. Under all conditions, the release of ACh from each of the 3 brain structures correlated with the level of locomotor activity.