We studied the effects of some chelators on creatine kinase activity. Creatine kinase is competitively inhibited by endogenous polyvalent cations (e.g., calcium, Ki = 4.5 mmol/L); this can be reversed by adding chelators to the reagent, resulting in a mean increase in activity of 1.14-fold at 25 degrees C and 1.18-fold at 30 and 37 degrees C. Adding chelators, 5 and 10 mmol/L, to serum stored at 37, 30, 25, 4, and -20 degrees C increased isoenzyme stability in some cases, but under certain conditions decreased it, especially at higher chelator concentrations, and more so for EGTA than for EDTA. Blood sampling into tubes prepared with chelators and storage of plasma has no advantage over serum stored in the presence of chelators. The most striking effect of chelators is their protective effect on thiols in the creatine kinase reagent. In the presence of EDTA, 2 mmol/L, the reagent is stable for at least a day at 25 degrees C or a week at 4 degrees C. The poor stability of glucose-6-phosphate dehydrogenase, which is nearly independent of chelators, is the limiting factor for reagent containing EDTA. Bis-Tris, a buffer recently recommended for assay of creatine kinase activity, is a weak chelator. Imidazole acetate buffer combined with EDTA yields activities identical to those found with Bis-Tris at assay temperatures of either 25 or 30 degrees C.