The m5C content was determined in three functionally different fractions of rat liver DNA isolated by a modified phenol fractionation. The transcriptionally active DNA-I contains up to 12-14% of hybrid RNA not hydrolyzed by RNAase, is enriched with unique sequences and makes up to about 20% of total DNA. In its total nucleotide content (43 mol.% of GC-pairs) DNA-I does not differ from the major fraction of DNA-II making up to about 70% of the genome, and from hardly extractable DNA-III, whose content in total DNA does not exceed 10%. However, the degree of methylation of the transcriptionally active DNA-I (1.28 mol.% of m5C) is higher than that of DNA-II and DNA-III (1.0 mol.% of m5C). Under cortisol-induced activation of transcription the number of repeating sequences in DNA-I is increased by about 10% concomitant with a decrease of these sequences in DNA-III. The hormone-induced changes in the transcriptionally active fraction of DNA are reversible. 4 hrs after cortisol injection a demethylation of highly repeating and supermethylation of normally repeating sequences in DNA-I are observed. The level of methylation of the unique sequences in DNA-I (Cot greater than 200) remains unchanged. The degree of methylation of various subfractions of nucleotide sequences of DNA-II and DNA-III afer cortisol injection does not differ from normal. Presumably the hormone-induced supermethylation of normally repeating sequences may control the transcription.