Stretch of smooth muscle can induce contraction and has been implicated in the aetiology of preterm labour, although few direct studies of its effects have been made. Multiple pregnancies (twins and higher) are at high risk of premature delivery. Progesterone significantly inhibits myometrial contractility by both genomic and non-genomic mechanisms. Large scale studies have confirmed a tocolytic (relaxant) benefit of progesterone in singleton pregnancies. In multiples however, similar studies have shown no such benefit. The reason for this difference is unknown. Our aims were therefore (1) to investigate, under controlled in vitro conditions, how myometrium from singleton and multiple pregnancies responds to different doses of progesterone and (2) how stretch affects the response of myometrium to progesterone. Myometrial strips were prepared from biopsies obtained with informed consent from 9 women with multiple pregnancy and 8 singletons undergoing Caesarean section at term. Once stable contractions arose, cumulative doses of hydroxyl-progesterone from 1 to 100µM were applied. In humanely killed term pregnant rat myometrium (n=5) stretches up to 2.5 fold relative to high-K induced maximal force, were applied with and without 100µM of Progesterone. In both rat and human studies, the strips were attached to a force transducer, superfused with physiological saline solution, and maintained at 37oC. A striking difference between multiples and singletons was found in the progesterone dose-response curves, with significant resistance to progesterone’s effects being found in the former; thus at 10µM progesterone myometrium from multiples produced 63±4% of control amplitude, but singletons produced only 29±14% of control force, mean ± sem Student’s T-test P=0.0063). Progesterone was able to reduce contractile amplitude at all levels of stretch examined, but its effects were significantly attenuated at higher levels of stretch. These preliminary results suggest significant differences in the myometrium in multiple pregnancies in its response to progesterone and that stretch may underlie this. The reduced efficacy of progesterone in multiple pregnancies may be due to the increased stretch accelerating the switching in progesterone isoforms in preparation for labour, (to a non-functional form), but leaves open the question of whether higher progesterone doses would be efficacious in preventing preterm delivery.