Different levels of damage and repair to eu- and heterochromatic DNA from the livers of rats receiving a dose of 10 mg/kg N-nitrosodimethylamine (NDMA) were apparent. Preincorporated ³H~thymidine was lost rapidly from euchromatic DNA but persisted in the heterochromatic fraction. Persistent damage, determined as single-stranded regions binding 10 benzoylated DEAE-cellulose (BD-cellulose), was evident in heterochromatic DNA for up to three months. By subjecting rats treated with NDMA to partial hepatectomy, generation of single-stranded regions in the newly synthesized heterochromatic DNA could be demonstrated. Such structural defects were evident when hepatectomy was performed two months after administration of the carcinogen. These findings indicate that structural damage to non-transcribed DNA is one of the most persistent molecular lesions following exposure to nitrosamines.