N-Methyl-N-nitrosourea (MNU) is a potent carcinogen in various sites of experimental animals and induces thymic lymphoma in rats, which has long been hard to induce by any carcinogen. To analyze the action of MNU on thymocytes, DNA strand breaking in thymocytes from the MNU-treated rat and that in MNU-treated cultured thymocytes were assayed. Fluorometric analysis of DNA unwinding (FADU assay), first reported by Birnboim and Jevcak to detect X-ray-induced DNA damage, was modified and applied to detect DNA damage in thymocytes treated with MNU in vitro or in vivo. In the present modified method, cell lysate was admixed with 0.15 M sodium hydroxide, and DNA unwinding was processed at pH 12.0 for up to 2 h at 0 degree C in iced water. Double-stranded DNA remaining after alkaline reaction was detected by binding ethidium bromide and measuring its fluorescence. The severity of DNA damage, both in vivo and in vitro, depended on the MNU concentration. In addition, the sequential survival rate and cell-size distribution of thymocytes treated with MNU in vitro were investigated. A close relationship between the severity of DNA damage and cell death was demonstrated in MNU-treated thymocytes, and DNA damage by a non-cell-killing dose of MNU was detected with this FADU assay. MNU-induced cell death is not programmed as in apoptosis, which is caused in thymocytes physiologically, immunologically and by X-ray irradiation or corticoids.