The activity of the transactivation functions (activation function (AF)-1 and AF-2) of the estrogen receptor alpha (ERalpha) is cell-specific. This study aimed to decipher the yet unclear mechanisms involved in this differential cell sensitivity, with particular attention to the specific influence that cell differentiation may have on these processes. Hence, we comparatively evaluated the permissiveness of cells to either ERalpha AFs in two different cases: (i) a series of cell lines originating from a common tissue, but with distinct differentiation phenotypes; and (ii) cell lines that undergo differentiation processes in culture. These experiments demonstrate that the respective contribution that AF-1 and AF-2 make toward ERalpha activity varies in a cell differentiation stage-dependent manner. Specifically, whereas AF-1 is the dominant AF involved in ERalpha transcriptional activity in differentiated cells, the more a cell is de-differentiated the more this cell mediates ERalpha signaling through AF-2. For instance, AF-2 is the only active AF in cells that have achieved their epithelial-mesenchymal transition. Moreover, the stable expression of a functional ERalpha in strictly AF-2 permissive cells restores an AF-1-sensitive cell context. These results, together with data obtained in different ERalpha-positive cell lines tested strongly suggest that the transcriptional activity of ERalpha relies on its AF-1 in most estrogen target cell types.