Mercaptopyruvate sulfurtransferase (MPST) plays a central role in both cysteine degradation and cyanide detoxification. Moreover, deficiency in MPST activity has been suggested to be responsible for a rare inheritable disorder known as mercaptolactate-cysteine disulfiduria (MCDU). To date, no mutation of the human MPST gene has been reported. We developed a screening strategy to search for mutations in the MPST gene of 50 unrelated French individuals. Two intronic polymorphisms (IVS1-110C>G and IVS2+39C>T) and a nonsense mutation (Tyr(85)Stop) were identified and their functional consequences were assessed in vivo by measurement of erythrocyte MPST activity and/or in vitro using heterologous expression or transient transfection assay. The nonsense mutation likely leads to the synthesis of a severely truncated protein without enzymatic activity, as supported by our in vitro data. This work constitutes the first report of the existence of a functional genetic polymorphism affecting MPST and should be of great help to investigate certain disorders such as MCDU.