Physiological concentrations of the pineal hormone melatonin inhibit the proliferation of estrogen-dependent (MCF-7), but not estrogen-independent (MDA-MB-330) breast cancer cells. Although both cell lines express low levels of the G protein-coupled mt1 melatonin receptor, higher levels of receptor are expressed by MCF-7 cells. To determine if the mt1 receptor mediates melatonin's growth-suppressive effect we examined several parameters m MCF-7 and MDA-MB-330 breast cancer cells stably-transfected with, and overexpressing the mt1 receptor (mt1-MCF-7, mt1-MDA). Using these transfected cells, a 46% enhancement of melatonin's growth-suppressive effects was seen in mt1-MCF-7 cells, however, MDA-MB-330 cells remained insensitive to melatonin's growth-inhibition. The mtl receptor agonist and antagonist demonstrated that these growth-inhibitory effects of melatonin in MCF-7 cells were mtl specific. As well, mt1 overexpression elicited MCF-7 cells supersensitive to melatonin's gene-modulatory effects. Melatonin enhanced pS2 expression in mt1-overexpressing cells by 145%, and significantly inhibited expression of ERalpha We then examined coupling of the mt1 receptor in parental, and mt1-overexpressing MCF-7 cells. Pertussis toxin was used so determine if inhibition of G alphai function would lead to a block of melatonin's growth-inhibitory effects However, melatonin's growth-inhibitory effects were determined to be insensitive to pertussis toxin in both parental and mt1-transfected MCF-7 cells. Yet, melatonin did inhibit forskolin-induced cAMP accumulation in parental and mtl transfected MCF-7 cells. Taken together, these data suggest that although the Galphai proteins do not appear to be the mediators of melatonin's growth-inhibitory effects, melatonin inhibits cAMP accumulation, suggesting that multiple pathways are involved In nude mouse tumor studies using mtl-transfected MCF-7 cells, mt1-overexpression significantly decreased tumor burden and weight. This inhibition was enhanced by melatonin Taken together, these data demonstrate that the mt1 receptor mediates melatonin's growth-inhibitory effects in ERalpha-positive human breast cancer cells, and that overexpression of the receptor results in a melatonin supersensitive phenotype. However, this effect depends upon expression of ERalpha. These data also demonstrate that the mt1 receptor mediates melatonin's growth-inhibitory effects in vivo and overexpression of the receptor dramatically inhibits proliferation and sensitizes tumors to melatonin's growth-inhibitory effects