We report optimal conditions for assaying highly purified human lactate dehydrogenase isoenzymes with the lactate-to-pyruvate and pyruvate-to-lactate reactions, as they apply to human serum. Interconversion of results between reactions is not practicable. Measurements of lactate dehydrogenase in either reaction direction at 25, 30, or 37 degrees C can be equally reliable if the volume fraction and the resulting deltaA/min is small. However, for interinstrument and interlaboratory comparisons, results from the lactate-to-pyruvate reaction are more reliable.