We have examined the distribution of individual adducts in repetitive DNA sequences of rat liver in vivo after a single dose of the carcinogen N-hydroxy-2-acetyl-aminofluorene. Repetitive fragments [82, 125, 179, 225, and 370 base pairs (bp)] were isolated by digestion of hepatic DNA with HindIII restriction endonuclease (EC 3.1.23.21) and gel electrophoresis. As assayed by 32P postlabeling, no qualitative differences were observed between the DNA-bound metabolites in repetitive sequences and total DNA, but preferential binding to these sequences occurred. After 1 day of treatment, the amounts of N-hydroxy-2-acetylaminofluorene-induced adducts were found to be 13.8, 2.0, and 3.0 times higher in 179-, 225-, and 370-bp repeats, respectively, than in total DNA, while 82- and 125-bp repeats showed no differences. The relative distribution of individual adducts varied among the various sequences. After 9 days, all five sequences showed 1.3-1.7 times higher binding as compared to total DNA. In contrast, a random binding was observed when DNA reacted in vitro with an active metabolite, N-acetoxy-2-acetylaminofluorene. Taken together, these results suggest that the enrichment and differential excision of adducts in the repetitive DNA sequences may be a function of the nuclear organization of DNA. This application of the 32P assay constitutes a means to study the DNA damage and excision repair in vivo in chromatin structural components, including transcribed and nontranscribed multiple-copy genes, in a much more sensitive and precise way than has been hitherto possible.