Estrogen modulates expression and function of G-protein-coupled receptors. The goal of this study was to assess the effect of 17β-estradiol (10 nM) exposure for 1 (E1) or 6 (E6) days on density and function of hMT1 and hMT2 melatonin receptors expressed in Chinese hamster ovary (CHO) cells (CHO-MT1/CHO-MT2 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-[125I]iodomelatonin-binding sites in CHO-MT2 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 MT1 or MT2 receptors; however, it significantly attenuated the maximal inhibition of forskolin-stimulated cAMP formation induced by melatonin (0.01–1 µM) in CHO-MT1 cells. Melatonin stimulation of [35S]GTPγS binding to CHO-MT1 cell membranes was also attenuated following estradiol treatment. The inverse agonist luzindole reduced basal [35S]GTPγS binding in estradiol-treated cells but not in control CHO-MT1 cells, suggesting that estradiol promotes constitutive activity of MT1 melatonin receptors. We suggest that 17β-estradiol differentially affects MT1 and MT2 melatonin receptor functions, attenuates melatonin responses through activation of MT1 receptors, and increases the MT2 receptors density.