Abstract The persistence of 6-thioguanine-resistant (TG r) T-lymphocytes was investigated in Fischer 344 rats treated with N-ethyl- N-nitrosourea (ENU) using two schedules. Male rats, aged 3 months, were given i.p. injections containing a total of 0, 50 or 100 mg ENU/kg either as a single treatment (single-dose group) or divided among 10 weekly treatments (split-dose group). At 1, 3, 5, 10, 20, 30 and 50 weeks after the single-dose treatment, and 10, 20, 30 and 50 weeks after beginning the split-dose regimen, animals were assayed for the frequency of TG r spleen lymphocytes. ENU produced significant dose- and time-dependent responses in the single- and the split-dose treatment groups. Although a few of the 50 mg/kg split-dose treatments were significantly higher than the comparative single-dose groups, the number of TG r lymphocytes produced by the two dosing regimens were generally similar. The frequency of TG r cells for control animals increased with the age of the animals. The mode of ENU administration did not greatly influence the percent cloning efficiency (%CE) of the non-selection cultures, although the %CE declined in animals over 10 months of age. To investigate the relationship between the frequency of TG r cells and the age of the animals at the time of ENU administration, additional rats aged 17 months were treated with a single dose of ENU and at 1, 5 and 10 weeks following exposure, the frequencies of TG r cells were determined from the isolated lymphocytes. No difference in mutagen sensitivity between rats treated at 3 months of age and those treated at 17 months of age was detected at the time points evaluated. The data demonstrate the persistence of ENU-induced TG r T-lymphocytes in the rat and suggest that the dose and possibly the treatment schedule, but not the age of the animal at the time of treatment, affect the response.