Although calmodulin (CaM) interaction with estrogen receptor alpha (ERalpha) has been known for more than two decades, it is only recently that the molecular mechanism of CaM-mediated regulation of ERalpha is beginning to emerge. Others and we have identified a putative calmodulin binding site (P(295)LMIKRSKKNSLALSTADQMVS(317)) in ERalpha, at the boundary between the hinge and the ligand binding domain. ERalpha mutations affecting its association with CaM have been reported to generate high basal, estrogen-independent transactivation activity, indicating that the P(295)-T(317) sequence has an inhibitory function. Moreover, we found that a synthetic peptide (ERalpha17p: P(295)-T(311)) containing residues crucial for CaM binding exerts estrogenic effects on breast carcinoma cells. Finally, computer-aided conformational studies revealed that the CaM binding site might associate with a region located downstream in ERalpha (the beta turn/H4 region), this association likely resulting in an auto-inhibitory folding of the receptor. Thus, we propose as a hypothesis that CaM acts as a positive regulator by relieving this ERalpha auto-inhibition.