The gas H2S is produced by multiple intracellular enzymes, and is proposed to be an endogenous vasodilator in systemic arteries. In pulmonary arteries (PA), however, H2S (‘sulphide’) causes a biphasic contraction resembling hypoxic pulmonary vasoconstriction (HPV) the unique response of PA to hypoxia. Since sulphide metabolism is thought to be inhibited as ambient O2 levels fall, it is proposed that HPV is due to a rise in cellular [sulphide] (Olson et al, 2006). We assessed this hypothesis in rat intrapulmonary arteries (IPA) using small vessel myography and other approaches. RT-PCR showed the presence of the sulphide-synthesising enzyme cystathione γ lyase (CGL), but not of cystathione β synthase (CBS) in IPA. The CSE blocker propargylglycine (PPG; 1mM) had no effect (9 ± 10% inhibition, n=5) on HPV, whereas the CBS inhibitor hydroxylamine (HA; 100μM) and the CGL/CBS blocker amino-oxyacetate (AOA; 100μM) reduced HPV by 54 ± 7% and 93 ± 10% (p<0.05, n = 5 and 4, respectively). HA and AOA similarly inhibited (by 46 ± 11%, n = 4 and 95 ± 6%, n = 3, respectively, p<0.05) contractions caused by the superoxide-generating drug LY83583. The HPV amplitude was unaffected by the presence of the sulphide donors GSH and GSSG (both 1 mM), but was increased by 97 ± 20% (p<0.05, n = 6) by the sulphide donor cysteine (1 mM). Cysteine caused a sustained contraction in both IPA and mesenteric resistance arteries if applied in the presence of a small degree of PGF2α induced tone, whereas sulphide (300 μM) contracted pre-constricted IPA but relaxed mesenteric arteries. Both hypoxia and 300μM sulphide caused a depolarisation of the mitochondrial membrane potential, recorded using TMRE, in IPA. The effect of sulphide on Δψ was larger than that of hypoxia (n=5), and was transient (10-15 min duration). In 2/5 IPA, depolarisation was preceded by a brief hyperpolarisation, and followed by a more sustained hyperpolarisation. Both hypoxia and sulphide caused an increase in the NADH/NADP+ ratio, manifested as an increase in the ratio of autofluorescence (measured at 510 nm) observed at excitation wavelengths of 340 and 380 nm (340/380 ratio). These results indicate that application of H2S at a concentration which causes a response resembling HPV inhibits the electron transport chain at least as much as does hypoxia. Although mRNA for CGL but not CBS was present in IPA, HPV was blocked by the CBS inhibitor AOA but not by the CGL inhibitor PPG. Each of the 3 antagonists of sulphide synthesis tested also had similar effects on the contraction caused by hypoxia and LY83583, which as a source of superoxide is very unlikely to cause contraction by increasing cellular [sulphide]. Collectively, the data show that sulphide is more likely to mimic the effect of hypoxia in rat IPA than it is to mediate it.