Ribosomal DNA (rDNA) methylation was studied in various strains of mice. We used restriction enzymes that are sensitive to methylation and a cloned probe containing the transcribed spacer and part of the 18S and 28S gene. Strains C3H/He3, C57/B6-3, and AKR/J were found to have less than 9% of the rDNA methylated. In sharp contrast, Balb/c mice showed 30-50% of the Hpa II and Hha I sites to be methylated. Further study of the Balb/c DNA showed that there are three groups of rDNA sequences. In the first group, all the Hpa II and Hha I sites are almost completely unmethylated; in the second group these sites are all methylated (greater than 30 sites for each enzyme); in the third group most sites are methylated, but there are discrete hypomethylation sites. These hypomethylation positions are at similar sites for both Hpa II and Hha I and show a tissue-specific pattern. Comparison of AKR/J with Balb/c copy level showed that AKR/J had about 60% fewer rDNA genes. The rDNA methylation level might thus be correlated directly with the number of rDNA genes. Finally, analysis of F1 mice from a cross between Balb/c and AKR/J showed both low copy number and low methylation levels.