Pyruvate kinase (PK) is an enzyme of critical importance in the glycolytic pathway of the red cells, deficiency of which, whether congenital or acquired, results in a hemolytic anemia. Measurement of the enzyme is now rapid and simple utilizing preprepared substrate and reagents. Some caution must be taken, however, in both measurement and interpretation. Red cells must be separated from other blood cells, particularly granulocytes, since the PK of white cells is different from that of red cells and is present in much higher activity per cell. Sometimes the use of special techniques of measurement [low substrate concentration (phospho-enolpyruvate, known as PEP, 0.4 mM in place of 2 mM) or heating at 53 degrees C for 60 minutes, (to test thermostability)] are necessary to detect abnormal molecular characteristics of PK, particularly in the acquired form of deficiency. Evidence recently presented suggests that the acquired form of PK deficiency may sometimes be due to inhibitors which oxidize sulfhydril bonds in the enzyme or alter its substrate binding properties or other molecular characteristics.