The suprachiasmatic nucleus of the mammalian brain is thought to be the anatomical locus of circadian rhythms. To examine the functional organization of the suprachiasmatic nucleus in vitro with intact intercellular connections for a prolonged period, we have established an organotypic slice culture system using a roller-tube technique. Brain slices (400 microns in thickness) containing the bilateral suprachiasmatic nuclei, were obtained from newborn rats at four to seven days old and were maintained in vitro for more than three weeks. During this three-week period, the slices flattened to one to three cell layers and two tightly packed neuronal cell-masses (neuronal zones), with diameters of about 1 mm were formed, which were surrounded by a peripheral glial cell-dispersed zone. In the neuronal cell zones, peptides and their messenger RNAs were found cytochemically with characteristic patterns similar to the suprachiasmatic nucleus in the brain. In situ hybridization and immunocytochemistry showed that vasoactive intestinal peptide messenger RNA expressing and vasoactive intestinal peptide-immunoreactive neurons were detected predominantly in the ventrolateral part of the neuronal zones in the suprachiasmatic nucleus slice culture. Vasopressin messenger RNA-expressing and vasopressin-immunoreactive cells were localized in the dorsomedial neuronal zones near the ependymal cell zone. The distribution of cell bodies and fibers containing these neuropeptides and their messenger RNAs in the neuronal zones of suprachiasmatic nucleus organotypic slice culture were similar to that of the suprachiasmatic nucleus in vivo. This suggests that the suprachiasmatic nucleus in these organotypic slice cultures retains the biological characteristics of these cells in vivo as the cells did develop, form compact neuronal masses and did establish connections. To examine the possibility that suprachiasmatic nucleus neurons in slice cultures show a persistent rhythmic activity, we also measured the amount of vasopressin released into the culture medium. Sampling at 4-h intervals combined with enzyme immunoassay revealed that vasopressin concentration in the medium embracing suprachiasmatic nucleus slice cultures fluctuated with a period of approximately 24 h. The present findings suggest that the intranuclear neuronal networks of the suprachiasmatic nucleus are maintained in vitro for a long duration and that organotypic cultures of the suprachiasmatic nucleus produce and release bioactive substances in an oscillating manner. The suprachiasmatic nucleus in slice cultures may be useful for future analysis of circadian rhythms in vitro.