The affinity of 2 methylating agents, N-methyl-N-nitrosourea (MNU), a potent carcinogen, and dimethyl sulfate (DMS), a very weak or non-carcinogen, for specific structural or functional regions of DNA has been studied in an in vitro system using rat liver nuclei. The release of alkylated nucleotides from nuclei following limited nuclease digestion was measured. Under restrictive conditions, pancreatic DNase I preferentially digests DNA sequences active in RNA transcription while micrococcal nuclease digests spacer DNA between nucleosome cores. Nucleotides methylated by methylnitrosourea were preferentially released early during the digestions, suggesting a localization in both actively transcribing regions and spacer DNA. DMS alkylation, on the other hand, showed a random distribution in chromosomal DNA as measured by micrococcal nuclease and only limited accumulation in transcribing DNA.