In the present study, using the model of ERα-negative cancer cells B16K1 grafted subcutaneously into syngeneic ovariectomized immunocompetent mice, the combination of genetic and pharmacological approaches revealed that both nuclear (AF1/AF2-mediated) and membrane actions of ERα are required for the pro-tumoral effects of E2 while the sole activation of membrane signaling of ERα is not sufficient. The gene discussed is ESR1; the disease is cancer.