Diverse forms of life have evolved 24-hour or circadian timekeeping systems serving to coordinate internal biological events with the daily solar cycle. The generation of circadian rhythms by this timekeeping system ensures that internal processes occur at the appropriate time of day or night in relation to the environmental cycle and to other functionally-affiliated events. For mammals, endogenous oscillations in gene expression are a prevalent feature of oscillatory cells residing in the suprachiasmatic nucleus (SCN) and non-SCN tissues. To determine whether immortalized cells derived from the rat SCN (SCN2.2) retain the intrinsic rhythm-generating properties of the SCN, oscillatory behavior of the SCN2.2 transcriptome was analyzed and compared to that found in the rat SCN in vivo. In SCN2.2 cells, 116 unique genes and 46 ESTs or genes of unknown function exhibited circadian fluctuations for 2 cycles. Many (35%) of these rhythmicallyregulated genes in SCN2.2 cells also exhibited circadian profiles of mRNA expression in the rat SCN in vivo. To screen for output signals that may distinguish oscillatory cells in the mammalian SCN from peripheral-type oscillators, the rhythmic behavior of the transcriptome in forskolin-stimulated NIH/3T3 fibroblasts was analyzed and compared relative to SCN2.2 cells in vitro and the rat SCN in vivo. Similar to the circadian profiling of the SCN2.2 and rat SCN transcriptomes, NIH/3T3 fibroblasts exhibited rhythmic fluctuations in the expression of the core clock genes and 323 (2.6%) functionally diverse transcripts. Overlap in rhythmically expressed transcripts among these different oscillator models was limited to the clock genes and four genes that function in metabolism or transcription. Coupled with evidence for the rhythmic regulation of the inducible isoform of nitric oxide synthase (Nos) in SCN2.2 cells and the rat SCN but not in fibroblasts, studies examining the effects of antisense oligonucleotide-mediated inhibition of Nos2 suggest that the gaseous neurotransmitter nitric oxide may play a key role in SCN pacemaker function. Thus, our comparative analysis of circadian gene expression in SCN and non-SCN cells has important implications in the selective analysis of circadian signals involved in the coupling of SCN oscillators and regulation of rhythmicity in downstream cells.