We developed standards for creatine kinase (CK; EC 188.8.131.52) assays by expressing human CK cDNAs in COS cells. Cells were transiently transfected with full-length cDNAs for CK subunits M and B, individually and in combination; and subsequently, high concentrations of CK activity were present in the cell lysate (1.2 U/mg protein). These proteins exhibited the characteristic isoenzyme-specific electrophoretic mobilities for CK MM and BB isoenzymes. We also produced subforms of CK MM and MB, identical to the modified CK variants produced in plasma after muscle or myocardial injury, by mutating the cDNA for the CK M subunit to delete the carboxy-terminal lysine residue. When this construct was cotransfected with the normal cDNAs for CK M and B, five electrophoretically distinct CK isoenzymes were detected by nondenaturing electrophoresis: MM3, MM2, MM1, MB2, and MB1. These proteins retained 100% of their activity after storage of the cell lysates - 20 or 4 °C for 3 months.