Circadian rhythms in humans, rodents and other animals tend to change as they age. The molecular mechanisms underlying these age-related changes in circadian rhythms are yet to be elucidated. DNA methylation is a chemical modification of DNA structure and an important epigenetic regulator of gene expression. In addition, DNA methylation differences have been observed to be acquired in multiple tissues with age [1]. To investigate whether DNA methylation of clock genes changed with age, and contributed to circadian dysfunction in aged animals, we examined the methylation of eight clock promoters (Per1, Per2, Cry1, Cry2, Clock, Npas2, Bmal1 and Bmal2) in the stomach, kidney, striatum and spleen using a methylation-specific PCR assay (MSP) [2]. Young (3-months-old, n = 14) and old (24-months-old, n = 14), male C57BL/6 mice were housed at an animal facility on a 12-h light-dark cycle (lights on at 08:00; lights off at 20:00) and allowed to feed ad libitum. Animal treatment was in accordance with the National Institutes of Health (NIH) guidelines for animal experimentation. All mice were sacrificed at 09:00 and samples of stomach, kidney, striatum and spleen were collected for DNA extraction. Methylation frequencies of clock genes in different age groups were compared and analyzed using Pearson Chi-square tests. We compared eight clock genes, using Bonferroni correction for multiple comparisons. Significance levels were established at a P-value less than 0.00625. Partial methylation was present at the Cry1 promoter in all tissues examined, at the Per1 and Bmal1 promoters in the stomach, and at the Bmal2 and Npas2 promoters in the spleen. Regarding age related changes, methylation frequency decreased significantly in the old mice at the stomach Per1 promoter (p = 0.006), but was significantly increased in old mice at the Cry1, Bmal2 and Npas2 promoters of the spleen (p = 0.001; p = 0.006; p = 0.002, respectively). Actually, no methylation was detected in the spleen of young mice, while 11/14 old mice demonstrated methylation in at least one locus, suggesting the spleen may be particularly prone to age-related circadian dysfunction. Indeed, circadian rhythms of cell proliferation in the spleen are reduced in aged rats [3]. These results indicated that methylation of some clock promoters were affected with age at least in the spleen and stomach; in turn this may lead to dysregulation of clock expression and changes in circadian rhythms [4]. In conclusion, our data supports the hypothesis that DNA methylation plays a role in age-related circadian dysfunction.