Although millions of clinical laboratory enzyme assays are performed daily, not a single authoritative reference method or material has been accepted which assures the fundamental reliability and/or compatibility of these assays. The objective of this investigation is to develop optimal assays and materials for two enzymes of major clinical importance. These enzymes, aspartate-aminotransferase (AspAT, formerly GOT) and lactate dehydrogenase (LDH), are among the main diagnostic enzymes for the various cardiac diseases; and AspAT has been used exclusively to detect and follow the course of liver disease. At the Clinical Chemistry Laboratories of the New York State Department of Health we have studied extensively the problems involved in determining the activity of these enzymes, and we have prepared highly purified standards for LDH I and cytoplasmic AspAT from human red cells. We propose now: 1. To prepare highly purified enzyme standards of their isoenzymes, LDH V (liver LDH) and mitochondrial AspAT, from human sources; 2. To investigate the behavior of all these isoenzymes in kinetic assays, under various combinations of substrate, buffer, temperature, and pH, in order to identify the optimal assay methods; 3. To make these optimal materials and methods available in such a way as to facilitate interlaboratory standardization; and 4. In the light of this study, to assess the value of enzyme assay reagents and kits being used in clinical laboratories to determine AspAT and LDH.