Extracellular levels of acetylcholine were measured by the microdialysis method coupled to high performance liquid chromatography in the dorsal hippocampus of freely moving rats over a period of 24 h to examine whether the acetylcholine release in the hippocampus exhibited a diurnal variation. Spontaneous motor activity was simultaneously measured with an automatic animal activity monitor. The amount of acetylcholine collected per 20-min sample varied markedly, in a range from about 5 to 90 pmol. There appeared to be variations in the amount with a 2-4 h periodicity as well as an apparent diurnal periodicity. In all five rats studied, the overall mean value for the dark cycle (11.1-34.5, average 20.9 pmol/20 min) was significantly greater than that for the light cycle (5.1-21.3, average 12.3 pmol/20 min), showing a 70% average increase. Cross-correlation analysis performed between the amount of acetylcholine and the motor activity count for the animal during the sampling revealed a significant positive correlation coefficient in four rats studied. The present study demonstrates for the first time that the acetylcholine release shows a diurnal variation.