alpha-Cyano-beta-(1-phenylindol-3-yl)acrylate inhibited pyruvate transport into both liver and heart mitochondria approximately linearly with respect to its concentration until 65% inhibition was achieved. The extent of inhibition was dependent on the mitochondrial protein concentration. By extrapolation of plots of inhibition versus inhibitor concentration to total inhibition, or by mathematical analysis of the plots, the concentration of pyruvate transporter molecules per mg of protein was calculated to be approximately 100 pmol/mg for both heart and liver mitochondria, and the Ki about 7 nM. The data also suggest that pyruvate transport is rate-limiting for pyruvate oxidation by heart mitochondria in State 3, but not by liver mitochondria.