Reporter assay system that employ firefly and Renilla luciferases is widely used as a conventional and powerful tool for monitoring single gene expression. One important application of the reporter assay system is the simultaneous monitoring of two gene expressions in the same cell population because the expressions of many genes are regulated concurrently by transcription factors. However, even with the current system, more than one gene expression cannot be monitored simultaneously because one of two luciferases must be used as an internal control. To overcome the limitations of the reporter assay system, we have recently developed a novel reporter assay system, tricolor reporter assay system, which consists green- and red-emitting Phrixothirix luciferases as dual reporters and Renilla luciferase as internal control. In this system, activities of green and red luciferases can be calculated simultaneously by devising the emissions with an optical filter. We have successfully employed this system in analyzing the effects of clock gene products CLOCK, BMAL1 and retinoid receptor-related orphan nuclear receptorα (RORα) on the enhancer elements of Per1 and Bmal1 promoters. The results indicate that the RORα regulates bidirectionally Bmal1 (positively) and Per1 (negatively) transcriptions simultaneously in the same cell populations. This system could be easily developed for monitoring four or more gene expressions using additional luciferases and optical filters, and for monitoring real-time rhythmic expression profiles in vivo. [Jpn J Physiol 54 Suppl:S30 (2004)]