The receptors for neurokinin 1 (NK1-R), neurokinin 2 (NK2-R), and neurokinin 3 (NK3-R) are expressed and functionally active in the uterus, promoting strong contractions of the myometrium. Previously, we demonstrated that myometrial contractility activated by the NK-Rs is regulated by estrogen. In the current study, we furthered our investigations of the role of estrogen in the regulation of NK3-R-mediated myometrial contractility. Estrogen promotes both heterologous and homologous desensitization of NK3-R-mediated uterine contractility. In tissue obtained from estrogen-dominated rats (ovariectomized estrogen-treated rats and rats in estrus), the magnitude of uterine contractions decreased in response to consecutive additions of the NK3-R-selective agonist senktide. By addition of the fourth dose of agonist, the contractile response was routinely barely above baseline. In contrast, in tissue obtained from non-estrogen-dominated rats consecutive doses of senktide resulted in contractions of identical magnitude. The homologous desensitization was specific to the NK3-R, and the desensitization of the NK3-R-mediated response did not affect the magnitude or nature of uterine contractions in response to NK1-R or NK2-R activation. Furthermore, heterologous and homologous desensitization of NK3-R-mediated contractility is dependent upon the duration of exposure to estrogen. This complex mechanism appears to be important in intact tissue; capsaicin-mediated release of endogenous neuropeptides resulted in a desensitization of response to subsequent stimulation with senktide in estrogen-dominated uterine tissue.