Estrogen modulates expression and function of G-protein-coupled receptors. The goal of this study was to assess the effect of 17β-estradiol (10 n M) exposure for 1 (E1) or 6 (E6) days on density and function of hMT<sub>1</sub> and hMT<sub>2</sub> melatonin receptors expressed in Chinese hamster ovary (CHO) cells (CHO-MT<sub>1</sub>/CHO-MT<sub>2</sub> cells). This strain of CHO cells expressed both estrogen receptor alpha and beta mRNAs, as determined by RT-PCR amplification. 17β-Estradiol treatment did not modify the affinity of either receptor; however, it significantly increased the density of 2-[<sup>125</sup>I]iodomelatonin-binding sites in CHO-MT<sub>2</sub> cells. 17β-Estradiol treatment (1–6 days) did not affect the potency of melatonin to inhibit forskolin stimulation of cAMP formation through activation of either MT<sub>1</sub> or MT<sub>2</sub> receptors; however, it significantly attenuated the maximal inhibition of forskolin-stimulated cAMP formation induced by melatonin (0.01–1 µ M) in CHO-MT<sub>1</sub> cells. Melatonin stimulation of [<sup>35</sup>S]GTPγS binding to CHO-MT<sub>1</sub> cell membranes was also attenuated following estradiol treatment. The inverse agonist luzindole reduced basal [<sup>35</sup>S]GTPγS binding in estradiol-treated cells but not in control CHO-MT<sub>1</sub> cells, suggesting that estradiol promotes constitutive activity of MT<sub>1</sub> melatonin receptors. We suggest that 17β-estradiol differentially affects MT<sub>1</sub> and MT<sub>2</sub> melatonin receptor functions, attenuates melatonin responses through activation of MT<sub>1</sub> receptors, and increases the MT<sub>2</sub> receptors density.