Acetylcholine concentrations were determined in suprachiasmatic nucleus of rats sacrificed by irradiation of microwave, using radioimmunoassay. No significant rhythmicity was observed over a 24-h period in rats blinded for two weeks. When the light was given at 22.00 h (illumination schedule L:07.00-19.00 h) in intact rats, acetylcholine concentration increased 30 and 60 min after light on in suprachiasmatic nucleus, but not in the other control site. These results suggest that endogenous circadian rhythm of acetylcholine concentration is absent in suprachiasmatic nucleus, but light may affect acetylcholine concentration of this site.