Increased 16alpha-hydroxylation of estradiol has been shown to be associated with heightened cancer risk in estrogen responsive tissue. Certain types of human papillomavirus (HPV) are cofactors for cancer in the cervix, an estrogen sensitive tissue. We have demonstrated that estradiol and 16alpha-hydroxyestrone increased the number of cells positive for proliferating cell nuclear antigen in HPV immortalized keratinocytes, the in vitro correlate of the premalignant keratinocyte. These estrogens caused the abnormal proliferation and anchorage independent growth, which correlates with malignant conversion. Indole-3-carbinol, a phytochemical in cruciferous vegetables known to preferentially induce 2-hydroxylation with minimal effect on 16alpha-hydroxylation, markedly blocked the ability of estradiol to increase anchorage independent growth. The results indicate that 16alpha-hydroxyestrone increases the malignant phenotype of HPV immortalized keratinocytes. However, indole-3-carbinol will block this response.