The estrogen receptor (ER) plays a critical role in the development of hormone-dependent cancer. Since HMGA1, a member of the "high mobility group" proteins, is overexpressed in certain malignant cells, we investigated the interaction between these nuclear proteins. Transfection of the HMGA1 expression vector increased 2-fold the transcriptional activation of ERE containing promoter by E(2). Furthermore, the HMGA1 protein stimulated severalfold the binding of purified ER to the consensus ERE oligonucleotides in gel mobility shift assays and saturation assays. However, HMGA1 could not bind alone either to consensus or to modified EREs, and the minor groove binding drug distamycin A failed to prevent the synergism between ER and HMGA1. This could suggest that the binding of HMGA1 to DNA was not required for its stimulatory effect. Antibody supershift assays showed that HMGA1 was required for increased binding and suggest a protein-protein interaction between those factors. This was confirmed by pull down assay. These data show that HMGA1 acts in concert with the ER to regulate the expression of estrogen responsive genes through a mechanism that does not require direct binding to DNA. These observations may be relevant in malignant cells expressing both proteins.