Dispersed cells of rat suprachiasmatic nucleus were cultured for more than a month with chemically defined medium. Arginine vasopressin and vasoactive intestinal polypeptide in the culture medium showed robust circadian rhythms starting 24 h after the cell dissociation. The two rhythms had similar periods, with a phase-lead of the vasoactive intestinal polypeptide peaks to the arginine vasopressin peak of about 1 h. The two rhythms remained two weeks later, with both peaks appearing at almost the same time, suggesting the synchronization of the two rhythms. Significant differences in cell architecture were detected depending on precoating matrices of culture dishes, which did not affect the circadian rhythms of arginine vasopressin and vasoactive intestinal polypeptide. Antimitotic treatment at the beginning of the culture not only reduced the number, but also changed the type of glial cells developed. The treatment did not interrupt the synchronized arginine vasopressin and vasoactive intestinal polypeptide rhythms until day 31. Early appearance of circadian rhythms indicates that neural networks in the suprachiasmatic nucleus are not necessary for the synchronous release of arginine vasopressin and vasoactive intestinal polypeptide. Glial proliferation is not essential for the generation, expression and synchronization of arginine vasopressin and vasoactive intestinal polypeptide rhythms in the dispersed suprachiasmatic nucleus cell culture.