Oxytocin receptor (OTR) regulation has been extensively studied in uterine myometrium and endometrium. However, studies in the cervix are limited. The present studies utilized in situ hybridization and immunocytochemistry to localize OTR mRNA and protein distribution in cervices of nonpregnant ovariectomized (OVX) rats and examined the effect of combined and independent treatments with estradiol and progesterone on cervical OTR. Thirteen nonpregnant rats were bilaterally OVX under general anesthesia. At least 7 days later, the rats were exposed to one of four different treatments 24 h prior to necropsy: 1) estradiol (50 μg, n = 4); 2) progesterone (10 mg, n = 3); 3) both estradiol (50 μg) and progesterone (10 mg) (n = 3); 4) corn oil vehicle (n = 3). After 24-h estradiol treatment, OTR mRNA increased significantly (p < 0.05) in smooth muscle cells of the rat cervix as a result of increased copy numbers of OTR mRNA per cell as well as an increased population of OTR mRNA-positive cells. Progesterone alone had no effect on OTR mRNA expression; however, progesterone combined with estradiol significantly inhibited the up-regulation of OTR mRNA by estradiol alone. The increase of OTR mRNA in cervical epithelial cells was minimal in all situations. Intensity of cervical OTR immunostaining in both the epithelial cells and cervical smooth muscle cells was also elevated after estradiol treatment. The anti-rat OTR antiserum used for immunocytochemistry was validated by Western blot analysis. In conclusion, OTR and OTR mRNA were localized in smooth muscle cells and in epithelial cells of rat cervix. Estradiol-dependent activation of OTR gene expression and active OTR synthesis in smooth muscle cells account for the increased OTR level in rat cervix in vivo, in which progesterone acted as an antagonist of estradiol on OTR gene expression.