To reduce pre-term delivery, new pathways and drugs that reduce uterine contractility are of interest. Hydrogen sulfide (H2S) is produced in vivo from L-cysteine, by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CBE). At least two enzymes degrade H2S; thiosulfate sulphur transferase(TST) and Thiol S- methyltransferase. Thus H2S will be regulated within cells. NaHS, which releases a rapid bolus of H2S, reduces myometrial contractility. However it is not clear if an H2S-generating system is present throughout gestation or if more physiological modes of H2S production can affect contractility. We have therefore: (1) examined throughout gestation, the effects on myometrial contractility of GYY4137 (GYY), a novel compound that slowly releases H2S, (2) compared GYY effects to those of NaHS in rat and human tissues, (3) investigated effects on Ca signalling, and (4) determined the myometrial expression of enzymes governing tissue H2S levels. Methods: Myometrial strips from biopsies obtained, with consent, from women undergoing term caesarean sections or hysterectomy, or non-pregnant (NP), 14, 18 or 22 day (term) pregnant and labouring rats were studied. Effects on contractility in response to GYY (1nM-1mM, Cayman chemicals) and 1mM NaHS were examined. Immunohistochemistry and western blotting were used to examine the expression of CBS, CSE and TST. Results: There was a significantly greater inhibitory effect of GYY and NaHS in pregnant compared to NP rat and human myometrium. The inhibitory effects of GYY and NaHS on contractility increased throughout gestation; however, in labouring myometrium neither GYY nor NaHS could reduce contraction. Thus at day 14, 18, 22 day and labouring, 1 mM GYY reduced amplitude (100%, control) to 78 ±10%, 52±13%, 38 ±6% and 106±5%, n=6, mean + sem. In term pregnant human myometrium the reduction by 1 mM GYY was 40±16%, n=5, and by NaHS was 9±2%, n=7. GYY’s effects were dose dependent with an EC50 of 0.7 ± 2µM. Underlying calcium transients were decreased in the presence of GYY (amplitude and frequency, n=4, p<0.01, day 22). Immunohistochemistry showed CBS and CSE in human and rat myometrium (n=3-7) and preliminary Western blot data indicates increased expression in NP compared to term myometrium (n=4). TST was not found in the myometrium (n=4 pregnant human and rat). Conclusions: NaHS and GYY produce uterine relaxation in a dose-dependent manner. Their effects increased throughout gestation, perhaps due to changes in H2S removal rates, which we found not to be via TST. Our data suggest that H2S contributes to uterine quiescence until labour onset. GYY decreased Ca transients, suggesting it affects L-type Ca channels, perhaps via sulphydration of residues. These data suggest that H2S is an attractive target for therapeutic manipulation of human myometrial contractility and drugs such as GYY will be effective.