Abstract: Melatonin has been shown to bind to the MT1 G protein-coupled receptor (GPCR) in MCF-7 breast cancer cells to modulate the estrogen response pathway suppressing estrogen-induced estrogen receptor alpha (ERα) transcriptional activity, blunting ER/DNA binding activity and suppressing cell proliferation. In these studies we have examined the effect of melatonin on the transcriptional activity of the ERα and other members of the steroid/thyroid hormone receptor superfamily, namely, the glucocorticoid receptor (GR) and the retinoic acid receptor alpha (RARα). As with the ERα, melatonin represses ligand (dexamethasone)-induced activation of the GR. This effect of melatonin on ERα and GR is blocked by pertussis toxin (PTX) suggesting that melatonin's actions may be mediated via a PTX-sensitive Gαi protein. In contrast, melatonin potentiates the action of all-trans-retinoic acid on RARα transcriptional activation and enhances RARα/DNA binding activity, an action which is not PTX-sensitive. Expression of a dominant-positive Gαi2 protein, with which the MT1 receptor has been shown to couple, is able to mimic the effect of melatonin on ERα but not RARα transcriptional activation in breast cancer cells. This demonstrates that GPCRs can modulate the transcriptional activity of various steroid receptors in response to their ligand through activation of different G protein signaling pathways.