The kinetic properties of lactate dehydrogenase (LDH) isozymes have been determined at high enzyme concentrations. Spectrophotofluorometric assays revealed that the extent of substrate inhibition of LDH-1 and LDH-5 depends on enzyme concentration. At high enzyme concentrations, in the range of those that exist in most mammalian cells, no inhibition by pyruvate occurred. Pyruvate concentrations up to and including 20.0 millimoles per liter were used for each isozyme at 25 degrees and 40 degrees C at pH 7.0 and 7.4. These results suggest that substrate inhibition of LDH may not occur in vivo but only in vitro after appreciable dilution from physiologic enzyme concentrations. These experiments provide further evidence against the theory that substrate inhibition of LDH-1 in vivo accounts for the distribution of LDH isozymes within various tissues. They raise the possibility that, for other enzymes, kinetic properties determined at highly dilute concentrations in vitro may also be quite different from kinetic properties at the much higher concentrations that exist in vivo.