Temporal patterns of release of aspartate, glutamate and glycine, which are related to excitatory amino acidergic transmission, were examined in organotypic slice cultures of rat suprachiasmatic nucleus over a 60 h period. Vasopressin release in the same culture was measured simultaneously to compare the temporal pattern with that of the amino acids. Amino acids and vasopressin were measured by high performance liquid chromatography and enzyme immunoassay, respectively. Robust circadian rhythms were detected in release of aspartate, glutamate and glycine. Glycine levels were about 10 times higher than those of aspartate and glutamate in the culture. Vasopressin also showed a clear circadian rhythm and the phase angle difference between each amino acid and AVP was not significantly different. The results indicate that cultured SCN cells release these amino acids and the release is under the control of the circadian pacemaker.