An age-related increase in DNA methylation of the multicopy 18S and 28S ribosomal RNA genes was found in CBA/Ca mice beginning between 6 and 18 months of age at the 5' end of these genes in liver, brain and spleen. The highest level of age-associated hypermethylation was mapped to the proximal 5' spacer domain. Silver staining of actively transcribing ribosomal genes in metaphase chromosomes from stimulated spleen cells provided cytological evidence that these mice have 3 rRNA cistrons located on chromosomes 15, 16, and 18. The ribosomal gene cluster located on chromosome 16 was preferentially inactivated in older animals. Exposure of spleen cells from older individuals to 5-azacytidine appeared to both reactivate ribosomal gene clusters and reduce rRNA gene methylation.