A specific labeling by radioactive N-ethylmaleimide of a protein involved in phosphate transport was obtained by protecting one of the two-SH-groups of the transport system with low concentrations of mersalyl. Subsequently, the other free-SH groups were blocked with excess N-ethylmaleimide. Removal of mersalyl by cysteine and subsequent inbucation with labeled N-ethylmaleimide results in a "specific" binding of N-ethylmaleimide to one-SH group functionally involved in phosphate transport. The isolated inner membrane fraction of the labeled mitochondria was subjected to dodecylsulfate gel electrophoresis. The followin results were obtained. 1. The difference of the radioactivity pattern on the dodecylsulfate-polyacrylamide gel of inner membrane proteins, labeled with N-[14C]ethylmaleimide in the absence and with N-[3H-A1-ethylmaleimide in the presence of mersalyl during preincubation of mitochondria, shows only one main labeled peak. The same labeled peak is obtained from the difference of labeling after preincubation with a constant low concentration of mersalyl at 32 degrees C and at 0 degrees C. 2. The position of the labeled peak on the dodecylsulfate-polyacrylamide gel corresponds to a protein of molecular weight of 26500 +/- 800. 3. The amount of one of the two-SH groups, involved in phosphate transport, was estimated to be 30 nmol per g of mitochondrial protein.