We previously reported that human natural autoantibodies enriched in antiestrogen receptor Ig (IgGs) display estrogenic activity in MCF-7 mammary carcinoma cells. In this study, we investigated IgGs' mechanism of action. We showed that: 1) IgGs Fab fragments (which contain only one antigen binding site) induced an estrogenic response in MCF-7 cells, producing estrogen receptor (ER) down-regulation and an increase in progesterone receptor concentration; 2) IgGs specifically inhibited MCF-7 cell surface labeling with fluorescent estradiol (E2)-BSA conjugates; 3) this inhibition of E2-BSA binding to membrane estrogen binding sites was largely caused by natural anti-E2-BSA antibodies (Ab) selectively associated with the natural anti-ER Ab within IgGs; 4) furthermore, these natural anti-E2-BSA Ab accounted for most of IgGs estrogenic activity in cell culture; 5) however, when incubated with cytosolic ER, they did not behave like estrogens, but they decreased ER hormone binding capacity; and 6) although IgGs stimulated cAMP production, their anti-E2-BSA Ab subpopulation did not. In conclusion, the estrogenic activity of IgGs does not involve Ab mimicking E2 molecular configuration or ligand-independent cAMP mediated pathways, membrane Fc receptors, and membrane receptor cross-linking mechanisms. On the contrary, IgGs seem to function by neutralizing estrogen-like epitopes, associated with ER-related peptides, which might inhibit ER activation.