The immature mammary glands of BALB/c female mice were treated with 7,12-dimethylbenz[a]anthracene (DMBA), 2 micrograms/ml, or 3-methylcholanthrene (10 micrograms/ml) for a 24-hr period at different times during the inital six days of lobuloalveolar growth in hormone-supplemented organ culture. Nodule-like alveolar lesions (NLAL) were detectable in 80% of the glands treated with DMBA (40% in 3-methylcholanthrene-treated glands) in the presence of insulin + prolactin + aldosterone + cortisol in the medium. No NLAL were present in dimethyl sulfoxide-treated control glands cultivated with the same hormones. The hormone combination insulin + prolactin + cortisol was unfavorable for NLAL induction by DMBA, and the combination of aldosterone + insulin + prolactin was only moderately conducive. Thus, the presence of cortisol with insulin + prolactin + aldosterone enhances NLAL incidence of mammary cells by DMBA. The highest incidence was found in glands that were treated with DMBA for 24 hr between the third and fourth day of culture, the period corresponding to the onset of the second wave of DNA synthesis in the gland. Cytotoxicity of DMBA was pronounced between 24 and 48 hr, when a high frequency of cells were in DNA synthesis, and survival of the cells after the cytotoxic effect of DMBA appeared to play a role in NLAL incidence. This suggests that DMBA-induction of NLAL in mammary glands in organ culture involves a complex carcinogen-hormone-cell cycle interaction. We emphasize that, although NLAL morphologically resembles the hyperplastic alveolar nodules of mouse mammary gland in vivo, the abilitity of NLAL to produce typical hyperactive alveolar outgrowth and mammary tumor after transplantation iv vivo remains to be determined.