N-ethylmaleimide (NEM) inhibits the transport of phosphate in mitochondria but is without effect on permeation of other metabolities. In spite of its specificity for inhibition of phosphate transport, NEM reacts in an unspecific manner with inner membrane proteins in general. Treatment of mitochondria with [3H]NEM just sufficient to produce inhibition of phosphate transport results in labeling of at least 10 polypeptide components of the inner membrane. A marked increase in the specificity of reaction of NEM for components of the phosphate transport system is attained by first protecting the transport system with p-mercuribenzoate (p-MB) and then by irreversibly blocking reactive sulfhydryl groups unassociated with transport by the addition of unlabeled NEM. Subsequent addition of dithiothreitol removes p-MB and restores 65 to 75 percent of the original phosphate transport activity. Reinhibition of transport with [3H]NEM results in both a 6-fold decrease in the amount of [3H]NEM bound by purified inner membrane vesicles and a substantial reduction in the number of labeled polypeptide components. Five distinct labeled species are detected by this method, one of which is a 32,000 molecular weight protein containing 40 percent of the bound radioactivity, or approximately 160 pmol/mg of inner membrane protein. Correlation of binding of [3H]NEM by inner membrane proteins with inhibition of phosphate transport suggests that the maximum concentration of the NEM-sensitive component of the phosphate transport system is 60 pmol/mg of mitochondrial protein. This value, when combined with V-max of NEM-sensitive transport of 205 nmol times min-1 times mg-1 at O degrees (Coty, W. A., and Pedersen, P. L. (1974) J. Biol. Chem. 249, 2593) yields an approximate minimum turnover for this process of 3500 min-1 at 0 degrees. This turnover number is at least 20-fold greater than similarly calculated values for adenine nucleotide transport and succinate oxidation in rat liver mitochondria at this temperature. Taken together these results suggest that the NEM-sensitive phosphate transport system in rat liver mitochondria has an unusually high catalytic activity compared to other mitochondrial processes, and that at least one of the five NEM-binding proteins is likely to be an essential component of this transport system.