Hydrogen sulphide (H₂S) is a gas signalling molecule which relaxes pregnant rat uterus. H₂S can be produced endogenously from L-cysteine by two enzymes, cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). The aim of the study was to investigate the production of H₂S from potential donors by intrauterine tissues in vitro. Tissue was obtained from pregnant rats and human placenta, with ethical approval. Homogenates of rat placenta, uterus, fetal membranes and human placenta were incubated with either L-cysteine (10 mM) or L-cystine (2 mM) or homocysteine (10 mM) (n = 9-10). Rat liver was used as a positive control and time-matched controls were run. H₂S production was measured as nM/min/g wet tissue using a standard method. With L-cysteine, rat placenta, uterus, fetal membranes and human placenta all produced H₂S. The order of production (mean nM/min/g) was rat liver (209)>rat fetal membranes (142)>human placenta (55.8)>rat uterus (52.3)>rat placenta (14.9). All of the tissues except human placenta produced H₂S from homocysteine. Rat liver and human placenta, but not rat placenta produced H₂S from L-cystine. DLpropargylglycine (inhibitor of CSE) and hydroxylamine (inhibitor of CBS) significantly inhibited production of H₂S. Intrauterine tissues produced H₂S from donors, via CBS or CSE, as a potential signalling molecule in vitro.