A critical factor in the quantitation of mutation induction in vivo is the time interval between treatment and sampling. In order to study mutagenesis in the mammary epithelium, the cell type in which breast cancer arises, we have measured the manifestation time, the minimum time required for the maximum mutant frequency to be achieved, in this tissue. The F1LacZtransgenic mice (Muta™ Mouse × SWR) were treated with N-ethyl-N-nitrosourea (ENU) at 50 mg/kg for five consecutive days and then sampled at 1, 2, 4, 6, 9, or 12 weeks after the last treatment. The LacZ−mutant frequency reached a maximum at 4 weeks post-treatment and did not vary significantly thereafter. Dlb-1−mutations in the small intestine reached a maximum at 2 weeks after treatment and did not vary significantly thereafter. Since the stage of estrus cycle during carcinogen exposure influences the mammary tumor incidence and latency, it was expected that it would also affect mutation induction. To test this, F1LacZmice in the estrus or di-estrus stage were treated with an acute dose of 250mg/kg ENU and sampled 10–13 weeks post-treatment. No statistical difference between the two groups was found, indicating that the effect of estrus on carcinogenesis is not due to variation in the sensitivity of the stage of the mammary gland to mutation.