Hydrogen sulfide (H2S) has recently been identified as an endogenous redox active gaseous mediator and proposed as an endogenous regulator of nitric oxide (NO) bioavailability, vascular tone and inflammation. Novel slow release H2S donor (SRHD) molecules such as GYY4137 [1,2] have been shown to regulate blood pressure in experimental and genetically induced hypertension and to inhibit tissue damage, oedema, cell death and inflammatory signalling [3-5] in sepsis and arthritis [6]; conditions well known to involve perturbed mitochondrial function and oxidative stress. In this study we have synthesised a series of compounds containing a mitochondria-targeting moiety (triphenylphosphonium), a C2-C18 ‘linker’ and H2S releasing moieties containing either a dithiolethione or thiohydroxybenzamide ‘tail’ (mitochondria-targeted SRHDs; mtSRHDs). To investigate H2S-mediated cytoprotection in vitro, we exposed human microvascular endothelial cells (HMEC) to oxidative injury with H2O2, 4-hydroxynonenal (4-HNE) and SIN-1 (or a cocktail of oxidants) in the presence or absence of either mitochondria-targeted H2S donors (< 100nM) or non-targeted SRHDs (GYY4137, AP67, AP72; < 200µM) for comparison. Oxidant induced cellular toxicity was assessed using standard metabolic assays, mitochondrial membrane depolarisation assays, ATP content, phosphatidylserine externalisation, caspase activation and activity etc. Intracellular oxidant production was estimated using intracellular fluorescent probes, DCF-DA, mitosoxRed and dihydroethidium. We also determined the effects on of SRHDs/mtSRHDs systemic blood pressure, heart and breathing rates in spontaneously hypertensive and L-NAME-treated rats. Preliminary experiments showed a C10 linker to optimal and AP39 (dithiolethione) and AP123 (hydroxythiobenzamide) were chosen for further study. Treatment of HMEC with SIN-1, H2O2 (100µM) 4-HNE (10µM) for 18h resulted in significant cytotoxicity (ANOVA,p<0.01). The toxicity of individual oxidants or oxidants used in combination to achieve similar effects (e.g. 50µM SIN-1/H2O2 with 2µM 4-HNE) was significantly inhibited by non-targeted SRHDs (100µM; p>0.01 all treatments). However, in each cytotoxicity assay the potency was substantially increased by targeting H2S delivery to mitochondria (AP39/123, 100nM; p<0.01). In spontaneously hypertensive and L-NAME (30mg/kg) treated rats, AP39 (1µM/kg) induced a rapid and sustained decrease in blood pressure and heart rate; an effect more pronounced than non-targeted SRHDs AP67 (30µM/kg) or GYY4137 (133µM/kg) [1]. These data suggest SRHDs can inhibit and / or reverse oxidative stress-mediated vascular cell injury and regulate vascular tone. Furthermore, strategies which increase H2S bioavailability, and in particular target mitochondria, represent a new therapeutic opportunity to limit mitochondrial and endothelial dysfunction.