SCN 2.2 cultures were stably transfected with luciferase reporter constructs driven by Ca(2+)/cAMP response element, E-box, or vasoactive intestinal peptide promoter to probe the circadian properties of this clock cell line. SCN 2.2 reporter lines displayed approximately 24-h rhythms of transcriptional activation after serum-shock. Serum-shocked cultures pulsed with glutamate exhibited phase-gated induction of phospho-CREB and of VIP, CRE, and E-box promoter activity. Glutamate-induced CRE promoter activity displayed restricted sensitivity to inhibitors of nitric oxide synthase and cGMP-dependent protein kinase. The temporal pattern of these sensitivities paralleled those of the SCN to light and glutamate during the night. Taken together, our data indicate that serum-shock can synchronize the circadian clock of SCN 2.2 cells to a state consistent with the day/night transition and, thus, establishes a temporal context for this cell line.