Treatment of mouse mammary glands with a high concentration of 7,12-dimethylbenzo(a)anthracene in whole organ culture was reported by Banerjee et al. to transform foci of lobuloalveoli to a hormone-independent state, and to give rise to mammary hyperplastic outgrowths and adenocarcinomas in vivo. In the present study using the identical system, mammary glands of BALB/c mice were exposed to 7,12-dimethylbenzo(a)anthracene or N-2-fluorenylacetamide at low concentrations that bring about maximal incidences of the hormone-independent hyperplastic lobuloalveolar lesions with minimal cytotoxicity. After morphological development of the lobuloalveoli in culture, the glands were enzymatically dissociated into cells and inoculated into gland-free inguinal mammary fat pads of syngeneic mice bearing pituitary gland implants during the initial 8 weeks. After 11 months, fragments of the resultant mammary outgrowths from each mouse were implanted into the gland-free inguinal mammary fat pads of 3 syngeneic mice (not bearing pituitary gland supplements) and were permitted to grow for another 11 months. Mammary outgrowths from the primary and secondary implants were neither neoplastic, anaplastic, nor dysplastic. Also, no hyperplasia in any mammary outgrowth could be attributed to the action of either carcinogen, especially when outgrowths were compared with contralateral outgrowths that arose from the control glands exposed to dimethyl sulfoxide (solvent of the carcinogens) in culture and/or with untreated thoracic mammary glands of the same hosts. One interpretation of these findings is that the hormone-independent, hyperplastic alveolar lesions may not be an appropriate in vitro marker of oncogenic transformation by chemical carcinogens in culture. The great variety of procarcinogens and activated carcinogens that bring about this lesion in vitro and its morphological similarity to presumptive mammary preneoplastic lesions in vivo weigh against this interpretation. A second hypothesis is that high concentrations of procarcinogens, despite their considerable cytotoxicity, complete a multistep process of oncogenic transformation in surviving mammary epithelium, whereas low concentrations optimized to produce the lesions in maximal number do not.