A large portion of the genome represents repetitive elements. Identifier (ID) elements, the major elements of short interspersed repetitive elements, are widespread with about 150 000 copies in the rat genome. Each I D element contains six CpG dinucleotides, which might account for the global methylation status of rat. We validated the CpG methylation of the ID elements by various methods. The methylation of one CpG site (CpG-3) of the ID element was investigated by performing pyrosequencing. The methylation percentage of the CpG-3 site was 53.6% (SD = 2.2) on average from six rat tissues with blood, but 24.6% (SD = 1.0) in rat pheochromocytoma, PC-12, cell line. This CpG-3 methylation was further verified by whole genome amplification (WGA), 5-azacytidine treatment, and proportional mixing of rat WGA genomic DNA (gDNA) with liver gDNA. Methylation-sensitive restriction enzyme PCR method showed that three other CpG sites (CpG-1, CpG-4, and CpG-5) within the ID element were also methylated (about 60%) in rat gDNA, but not in WGA gDNA. The ID elements may be good candidates for routine analysis of the global DNA methylation changes of rat for pharmaceutical treatment and their use can make basic epigenetic research possible with high accuracy.