Nitroxyl (HNO) displays distinct pharmacology to its redox congener nitric oxide (NO*) with therapeutic potential in the treatment of heart failure. It remains unknown if HNO donors are resistant to tolerance development following chronic in vivo administration. Wistar-Kyoto rats received a 3-day subcutaneous infusion of one of the NO* donors, glyceryl trinitrate (GTN) or diethylamine/NONOate (DEA/NO), or the HNO donor Angeli s salt (AS). GTN infusion (10mug/kg/min) resulted in significantly blunted depressor responses to intravenous bolus doses of GTN, demonstrating tolerance development. By contrast, infusion with AS (20mug/kg/min) or DEA/NO (2mug/kg/min), did not alter their subsequent depressor responses. Similarly, ex vivo vasorelaxation responses in isolated aortae revealed that GTN infusion elicited a significant 6-fold decrease in the sensitivity to GTN and reduction in the maximum response to acetylcholine (ACh). Chronic infusion of AS or DEA/NO had no effect on subsequent vasorelaxation responses to themselves or to ACh. No functional cross-tolerance between nitrovasodilators was evident, either in vivo or ex vivo, although an impaired ability of a nitrovasodilator to increase tissue cGMP content was not necessarily indicative of a reduced functional response. In conclusion, HNO donors may represent novel therapies for cardiovascular disease with therapeutic potential over clinically used organic nitrates.