Adolescent idiopathic scoliosis (AIS) represents the most frequently occurring form of scoliosis that occurs and progresses in puberty. This critical period coincides with many biological changes related to estrogens. The aim of this study was to determine the effect of 17-beta-estradiol on the responsiveness of AIS osteoblasts to melatonin and the cross-talk between estrogen and melatonin at the levels of the G(S)alpha and G(i)alpha proteins. Human osteoblasts derived from AIS (n = 40) and control patients (n = 10) were first screened for their functional response to the melatonin and 17-beta-estradiol. In response to the 17-beta-estradiol in a specific group of scoliotic patients, the level of 3',5'-cyclic adenosine monophosphate (cAMP) was significantly decreased when compared with the level observed in the presence of increasing concentrations of melatonin alone. Ours results provide strong evidence of the cross-talk between 17-beta-estradiol and melatonin signaling in human AIS osteoblasts. These results indicate a novel role for 17-beta-estradiol and melatonin in AIS, controlling the coupling of G(S)alpha protein and MT2 receptor on human osteoblasts. We found that the increased cAMP levels induced by melatonin can be corrected by the treatment of the cells with 17-beta-estradiol. Thus, estrogens or estrogen receptor agonists become important compounds to consider in AIS osteoblast cell functioning. Consequently, our results add a new facet to the understanding the role and function of melatonin in AIS.