Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM), two potent smooth-muscle relaxants, have been shown to cause uterine relaxation. Both CGRP- and AM-binding sites in the uterus increase during pregnancy and decrease at labor and postpartum. These changes in binding sites appear to be related to the changes in calcitonin receptor-like receptor (CRLR), receptor activity-modified protein 1 (RAMP₁), RAMP₂, and RAMP₃ mRNA levels. It is not clear, however, whether the changes in the receptor components occur in the myometrial cells and whether the steroid hormones can directly alter these receptor components in the muscle cells. In addition, the mechanism of CGRP and AM signaling in the rat myometrium is not well understood. Therefore, we examined the mRNA expression of CGRP- and AM-receptor components, G protein Gαs, CGRP, and AM stimulation of cAMP and cGMP, and the effects of progesterone on these parameters in the Eker rat uterine myometrial smooth-muscle cell line (ELT3). ELT3 cells expressed CGRP- and AM-receptor components CRLR, RAMP₁, RAMP₂, and RAMP₃. Expression of CRLR and RAMP₁ mRNA increased with progesterone treatment and decreased with estradiol-17β treatment. However, RAMP₂ and RAMP₃ mRNA expressions were unaltered by both progesterone and estradiol. Progesterone increased (P < 0.05) Gαs expression and augmented CGRP- and AM-induced increases in cAMP levels. In uterine smooth-muscle cells, the antagonist to Gαs protein NF449 decreased basal as well as CGRP- and AM-stimulated cAMP levels. None of the cell treatments affected cyclic GMP production. Our results suggest that the progesterone-stimulated increases in CGRP and AM receptors, Gαs protein levels, and cAMP generation in the myometrial cells may be responsible for increased uterine relaxation sensitivity to CGRP and AM during pregnancy.