The time between treatment and the appearance of mutants (mutant manifestation time) is a critical variable for in vivo transgenic mutation assays. There are, however, limited data describing the optimal sampling time for detecting mutations in various tissues of mutagen-treated animals. In this study, we investigated the time course of cII gene mutant induction in the liver, spleen, and bone marrow of Big Blue transgenic mice treated with N-ethyl-N-nitrosourea (ENU). Six-month-old female mice were treated with a single dose (120 mg/kg) of ENU, and the animals were sacrificed, and the cII mutant frequencies (MFs) were determined at 1, 3, 7, 15, 30, and 120 days after the treatment. The MFs in the liver cII gene of ENU-treated mice increased with time after the treatment, while the MFs for concurrent controls remained constant. The liver cII MFs in ENU-treated mice were significantly increased at day 30 and 120 (p < 0.01), with the largest increase at day 120. The spleen cII MFs in ENU-treated mice were increased significantly at day 7 and later (p < 0.01), and reached a plateau at day 30. In the bone marrow, the cII MFs in ENU-treated mice were increased significantly at all sampling times (p < 0.01), with the maximum MF at day 3. These results confirm that the time after treatment required to reach the maximum MF is tissue specific, with the approximate time for the maximum ENU-induced cII MF response being: bone marrow, 3 days; spleen, 14-30 days; and liver, more than 30 days.