The in vivo binding of radioactive N-2-acetylaminofluorene (AAF) and N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to the DNA of rat liver chromatin was examined. The chromatin was fractionated into putative transcriptionally active and inactive fractions by hydrodynamic shearing and subsequent glycerol gradient centrifugation, DNAase II digestion followed by MgCl2 aggregation of transcriptionally inactive chromatin, or mild digestion with micrococcal nuclease. Carcinogens were administered for various times prior to sacrifice. Irrespective of the duration of exposure, no preferential binding of either carcinogen to DNA was detected in any of the fractions prepared by hydrodynamic shearing of DNAase II digestion. When micrococcal nuclease was utilized, a 2-fold increase in carcinogen bound to the DNA of that chromatin fraction containing the smallest molecular weight fragments was detected. These small molecular weight fragments produced by micrococcal nuclease have been postulated to be derived from in vivo transcriptional units. Additionally, when DNAase II was used to probe chromatin from rat livers which had been exposed to a carcinogenic regimen of AAF, no preferential binding of radioactive N-OH-AAF to the DNA of any chromatin fraction was detected.