We investigated the biological significance of estrogen receptors (ER) in NIH3T3 cell transformation by the [12Val] K-Ras mutant. This mutant enhanced the steady state level of ER. Cells expressing mutant K-Ras (K12V cell) were tumorigenic. To determine the role of ER accumulation in Ras-transformed cells, we developed cells (KwtER cells) that overexpressed both wild-type (wt) K-Ras and ER, and found these cells were also tumorigenic. E2 stimulated the transcriptional activity by ER dominantly in K12V cells. However, only partial activation of ER by E2 was seen in KwtER cells. In the presence of 10% serum in media, the activation of ER appeared only in transformed KtwER and K12V cells, suggesting that two independently transmitted signals, the E2-ER binding and the ER-AF1 activation, are necessary for ER activation and that the dominant activation of ER might be involved in Ras-mediated cell transformation. Co-expression of progesterone receptor (PR) with mutant K-Ras led to suppression of tumorigenicity and inhibition of the activation of ER. The antisense oligomers complementary to the ER suppressed proliferation and transformed phenotypes of K12V cells. These observations support the importance of ER in Ras-mediated cell transformation.