RNA radioactively methylated with dimethyl sulfate has the advantage of relatively low background noise level when utilized in DNA saturation hybridization employing the membrane filter technique. In addition, the RNA can be methylated with either 3H- or 14C-labeled methyl groups. However, the low specific radioactivity usually obtained with dimethyl sulfate has limited the use of this labeling technique. We describe a detailed characterization of the methylation of rRNA with dimethyl sulfate giving specific radioactivities on the order 10-5 cpm/mug. Kinetics and optimum conditions for the methylation reaction of rRNA were studied. The methylation did not cause excessive degradation of RNA in neutral aqueous solution, and the methyl derivative of RNA was stable under normal hybridization conditions. Specific radioactivitiy of the methylated RNA was found to be a linear function of the product of RNA concentration and specific radioactivity of the dimethyl sulfate in the reaction mixture at a constant incubation time. The methylated bases of the RNA lowered the thermal stability of the DNA-RNA hybrids by 1 degree in Tm per 1.6 per cent methylated RNA bases. rRNA gene dosage values using high specific radioactive methylated RNA were found to be 81 and 180 genes/haploid genome, respectively. Dissociation constants of the hybridization reaction ranged from 0.90 times 10-10 to 2.37 times 10-10 M.