PTHrP has been localized in the uterine endometrium and myometrium while CGRP has been localized in nerves in the uterus. We have previously reported that both peptides relax uterine contraction in a dose-related manner using tissue from nonpregnant and pregnant rats. We have been interested in the signal transduction of these peptides in the uterus. ATP-sensitive K+ channels are present in various tissues including smooth muscle and have been suggested to play a role in the vasodilatory effect of CGRP in uterine arteries. Thus, we investigated the role of ATP-sensitive K+ channels and PTHrP, CGRP and ritodrine, a β-agonist, on the relaxation of rat uterine tissue. We used an in vitro uterine assay utilizing 3-10 tissue strips for each dose from 38 nonpregnant rats. PTHrP(5×10-7-10-9 M), CGRP (10-6-10-9 M), ritodrine (10-6-10-9 M) and cromakalim (10-4-10-7 M), an ATP-sensitive K+ channel activator, inhibited acetylcholine(Ach)-stimulated (10-6 M) uterine contraction in a dose-related manner. Glyburide (10-4-10-5 M), an ATP-sensitive K+ channel blocker significantly blocked the ability of cromakalim at all doses to inhibit Ach-stimulated uterine contraction. However, using the same paradigm, glyburide at effective doses failed to block PTHrP, CGRP and ritodrine inhibition of Ach-stimulated uterine contraction. These data suggest, that in our assay, cromakalim reactive ATP-sensitive K+ channels do not play a role in PTHrP, CGRP and ritodrine relaxation of Ach-stimulated uterine contraction.