The role of body fat as a risk factor for breast cancer has been well established. A decrease in the urinary 2/16α-hydroxyestrone ratio has also been shown to be a risk marker for breast cancer. These two observations are connected by the fact that obese women have decreased levels of 2-hydroxyestrone. To test the hypothesis that fat depots secrete factors that inhibit 2-hydroxylation, the effect of substances released into the media from adipocytes incubated in Krebs-Ringer buffer, on estrogen metabolism by MCF-7 cells in minimum essential medium eagle (MEM) plus adipocyte-conditioned media (ACM) was studied. The 1:1 ACM-MEM culture system resulted in a substantial and highly significant decrease in 2-hydroxylation of estradiol. This inhibition was partially reversed by the addition of indole-3-carbinol, a potent inducer of 2-hydroxylation of estradiol. Centrifugal sizing showed that the active 2-hydroxylation inhibitor in the medium had a molecular weight of about 30 kDa. These results suggest a mechanism for the decrease in 2-hydroxylation of estradiol that is observed in obese women and the increase in 2-hydroxylation observed in women with depleted fat depots.