Hydrogen sulfide (H2S) has rapidly emerged as an additional ‘gasomediator’ important in health and disease and recently compounds which generate H2S have been proposed as novel therapeutic agents. H2S can be synthesised in mammalian cells from cysteine/homocysteine via the PLP-dependent enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) in the cytoplasm. Mitochondria are also capable of generating H2S from mercaptopyruvate via 3-mercaptopyruvate sulfurtransferase (3-MST) where H2S is thought to stimulate mitochondrial respiration and ATP synthesis, and regulate cell death and inflammation. Oxidative stress results in the inhibition of mitochondrial H2S synthesis and/or depletion of mitochondrial H2S and the translocation of CSE and CBS to mitochondria resulting in mitochondrial H2S synthesis, cellular respiration and cell survival. Cellular and mitochondrial H2S levels are depleted, or H2S synthesis is inhibited, in vascular disorders such as hypertension, COPD, heart failure and diabetes etc. Pharmacological inhibition or genetic removal of CSE exacerbates the detrimental effects of oxidative stress (e.g. mitochondrial dysfunction), on inflammation, heart and blood vessel damage in atherosclerosis, hypertension and pre-eclampsia. Conversely, ‘H2S replacement’ with source of H2S such as sulfide salts (e.g. NaSH) or donors (e.g. GYY4137) prevents/reverses mitochondrial damage in these models. These studies have suggested that mitochondrial generation of H2S is crucial for cell survival and approaches to ‘replenish’ lost H2S offer a novel therapeutic approach for the treatment of disease. However, neither NaSH nor GYY4137 deliver H2S to mitochondria. We have therefore developed a series of novel mitochondria-targeted H2S donors (mtH2SD) containing a well characterised targeting moiety triphenylphosphonium, a variable length aliphatic linker and sulfide generating moieties e.g. hydroxythiobenzamide or anethole dithiolethione. In vitro experiments using mtH2SD in a variety of cells showed potent (e.g. <100 nM) cytoprotection against biological oxidants (e.g. H2O2, 4-HNE, peroxynitrite, glucose oxidase, hyperglycaemia etc) and abrogation of mitochondrial and cellular ‘ROS’ production, mitochondrial and cytoplasmic protein and DNA damage, ATP synthesis and cellular bioenergetics (Seahorse). From these studies two lead compounds were identified, AP39 (containing ADT-OH) and AP123 (containing HTB). In each assay AP39 and AP123 were shown to confer greater cytoprotection, reduced oxidative stress and mitochondrial damage than GYY4137 (or NaSH) at substantially lower concentrations (e.g. 30-100 nM c.f. 200 µM GYY4137). In NO-deficient (L-NAME) rats, AP39 lowered heart rate and systemic blood pressure and reversed arterial stiffening (180 µg/kg c.f. 25 mg/kg µmol/kg GYY4137). Patch clamping experiments showed AP39 inhibited T-type Ca2+ channels (< 300 nM) as well as RyR2 channels in isolated cardiac sarcoplasmic reticulum suggesting a mechanism of action that may include regulation of intracellular calcium levels. Recent data from in vivo studies using low doses of AP39 (e.g. 7 – 200 µg/kg) in models of cardiac arrest, myocardial and renal ischaemia-reperfusion will be discussed. These studies strongly suggest that targeting mitochondria with a source of H2S may represent a novel therapeutic approach to treat human disease.