Circadian disturbance of clock gene expression is a risk factor of diseases like obesity, cancer and sleep disorder. For the study of these diseases, it is necessary to monitor and analyze the expression rhythm of clock genes in the whole body for a long duration. The bioluminescent reporter enzyme firefly luciferase and its substrate D-luciferin have been used to generate optical signals from tissues in vivo with high sensitivity. However, little is known about the stability of D-luciferin to detect gene expression in living animals for a long duration. In the present study, we examined the stability of luciferin solution over 21 days. L-luciferin, which is synthesized by racemisation of D-luciferin, had a high concentration after 21 days. In addition, we show that bioluminescence of Period1 (Per1) expression in the liver was significantly decreased comparing with a 1 day solution, although locomotor activity rhythm was not affected. These results show that D-luciferin should be applied to the mouse within at most 7 days to detect the bioluminescence of Per1 gene expression rhythm in vivo.This article is protected by copyright. All rights reserved.