Recent clinical trials have demonstrated a beneficial effect of supplementation with progesterone to prevent preterm labor. We aimed to determine the effects of progesterone treatment in vitro and in vivo and 17alpha-hydroxyprogesterone caproate (17OHPC) in vitro on myometrial contractions.Methods: Myometrial strips were taken from women undergoing cesarean delivery at term. We also obtained myometrial biopsies from women participating in a clinical trial of progesterone to prevent preterm labor in twins (STOPPIT). After establishment of spontaneous contractions, strips were exposed to progesterone or 17OHPC. Separate strips were exposed to oxytocin and tocolytics alone and in combination with progesterone. Potassium channel blockers were added in conjunction with progesterone. STOPPIT samples were used to compare the effects of in vivo progesterone and placebo. We measured amplitude, frequency and activity integral of contractions. Results: Maximum inhibition of contraction amplitude was 93 +/- 2% and 67 +/- 14% for progesterone at 30 mu M and vehicle (70% ethanol), respectively, P <0.05. 17OHPC did not exert an inhibitory effect. Water soluble progesterone exerted a maximal inhibitory effect on amplitude of contractions of 82 +/- 10% at 100 mu M, P <0.05. The inhibitory effect of progesterone was unaffected by potassium channel blockers. There was no difference between in vivo placebo and progesterone-treated groups in either amplitude or frequency of contractions, nor was there any difference in the response to oxytocin or the tocolytic drugs. Conclusions: Progesterone exerts rapid inhibition of the amplitude of myometrial contractions in vitro but 17OHPC does not. The action of progesterone does not appear to operate via potassium channels nor does it enhance the activity of certain tocolytic drugs.