Free radical damage has the potential to significantly affect the behavior of cells in culture. In this study the effects of antioxidants (superoxide dismutase, catalase, and vitamin E) and lowered oxygen tension (1% oxygen) on primary culture of rat mammary epithelial cells were examined. Rat mammary epithelial cells were dissociated in collagenase with or without the addition of antioxidants and low oxygen tension, then cultured for 10 d in rat-tail collagen gel matrix and fed with Dulbecco's modified Eagle's F12 medium supplemented with various hormones and growth factors. Growth potential of the mammary cells was enhanced when antioxidants and low oxygen tension were used, alone or in combination, during the cell dissociation period. Using antioxidants and low oxygen tension during the culture period failed to improve growth potential regardless whether cells were dissociated in standard conditions or with antioxidants and low oxygen tension. The use of antioxidants and low oxygen tension during the cell dissociation period also reduced the degree of keratinization of the cells after 10 d of culture. Using antioxidants and low oxygen tension during the cell culture period did not further reduce keratinization if antioxidants and low oxygen tension were used during the dissociation period, but were effective in reducing keratinization if cells were dissociated in standard condition. In this system, antioxidants and low oxygen tension reduced lipid peroxidation during the cell dissociation period. An iron chelator, desferal, can also reduce lipid peroxidation and enhance growth when used during cell dissociation, suggesting the enhanced growth potential by the addition of antioxidants and low oxygen to be due to the reduction of lipid peroxidation.