The gene encoding parathyroid hormone-related protein (PTHrP), an autocrine/paracrine inhibitor of vascular and nonvascular smooth muscle contractility, is regulated by hormonal steroids including estrogens (E2), 1,25-dihydroxy vitamin D (Vit D3) and glucocorticoids. While E2 increases PTHrP gene expression, Vit D3 and glucocorticoids inhibit transcriptional activity of this gene. In the uterus of ovariectomized rats, E2-treatment increases both PTHrP mRNA levels and smooth muscle sensitivity to the action of PTHrP(1-34). To examine the action(s) of Vit D3 and glucocorticoids on these parameters, OVX rats were treated with E2, Vit D3 or the synthetic glucocorticoid, dexamethasone (Dex), alone, or with E2 following a 1 h pretreatment with Vit D3 or Dex. PTHrP and PTH/PTHrP receptor mRNA were measured by blot hybridization analysis of RNA prepared from uteri collected 2, 4 and 24 h after treatment. Uterine horns were used to measure the effect of the steroids on the ability of PTHrP(1-34) to inhibit spontaneous myometrial contraction. When E2, Vit D3 and Dex were given alone, only E2 altered PTHrP mRNA levels in the uterus, however, a 1 h pretreatment with Dex but not Vit D3 markedly diminished this effect of E2. The temporal decline in uterine PTH/PTHrP receptor mRNA levels measured 2 and 4 h after E2 treatment inversely correlated to changes in sensitivity of the tissue to PTHrP(1-34) measured at 24 h after E2 administration. In comparison to E2 alone, treatment with Vit D3 and E2 augmented the uterine responsiveness to PTHrP(1-34) while pretreatment with Dex (1 mg/kg) and E2 decreased this response. These data indicate that in the uterus, Dex opposes the positive effect of E2 on PTHrP gene activity and differentially modulates the action of PTHrP on myometrial tone. Moreover, elevations in the circulating levels of cortisol at term may serve to decrease both the uterine expression of PTHrP and the local action of PTHrP on the myometrium prior to parturition, therefore promoting myometrial contraction associated with labor.