The expression of two human estrogen receptor-(heR) isoforms has been characterized within ERa-positive breast cancer cell lines such as MCF7: the full-length heR66 and the N terminally deleted heR46 which is devoid of AF-1 transactivation function. Although heR66 is known to mediate the mitogenic effects that estrogens have on MCF7 cells, the exact function of heR46 in these cells remains undefined. Here, we show that, during MCF7 cell growth, heR46 is mainly expressed in the nucleus at relatively low levels, while heR66 accumulates in the nucleus. When cells reach confluence, the situation reverses, with heR46 accumulating within the nucleus. Although heR46 expression remains rather stable during an estrogen-induced cell cycle, its over-expression in proliferating MCF7 cells provokes a cell-cycle arrest in G0/G1 phases. To gain further details on the influence of heR46 on cell growth, we used PC12 ERa-negative cell line, in which stable transfection of heR66 but not of heR46 allows estrogens to behave as mitogens. We next demonstrate that, in MCF7 cells, over-expression of heR46 inhibits the heR66-mediated estrogenic induction of all AF-1 sensitive reporters: c-fos, cyclinD1 as well as ERE-driven reporters. Our data indicate that this inhibition occurs likely through functional competitions between both isoforms. In summary, heR46 antagonizes the proliferative action of heR66 in MCF7 cells in part by inhibiting heR66 AF-1 activity.