We have previously reported that spontaneous release of excitatory amino acids (aspartate and glutamate) show remarkable circadian rhythms in the organotypic slice culture of rat suprachiasmatic nucleus (SCN). Here we showed effects of extracellular Ca(2+) removal and of L-trans-pyrrolidine-2,4-dicarboxylic acid, a glutamate/aspartate uptake inhibitor on the circadian release of excitatory amino acids in the SCN culture. Amino acids were measured by high-performance-liquid-chromatography. Removal of extracellular Ca(2+) exerted no effect on the spontaneous release of the excitatory amino acids, while it blocked high K(+)-evoked release of the amino acids. Neither the period nor the amplitude of the spontaneous circadian release of amino acids in Ca(2+)-free medium was different from those in the Ca(2+)-containing medium. On the other hand, L-trans-pyrrolidine-2,4-dicarboxylic acid increased the excitatory amino acid levels without affecting the amplitude of excitatory amino acid rhythms. These results indicated that the circadian release of excitatory amino acids in the SCN is Ca(2+)-independent and L-trans-pyrrolidine-2,4-dicarboxylic acid- insensitive. Therefore, Ca(2+)-dependent chemical synaptic transmission may not be involved in the circadian rhythm generation in the SCN.