We studied a electrophoretic method for creatine kinase (CK, EC 2.7.3.2) MB sub-bands using a discontinuous buffer system on cellulose acetate membrane. The sub-bands (CK-MB1 and MB2) were clearly separated using a discontinuous buffer consisting of 0.22 mol/1 tris aminomethane-glycine buffer on the anode side and 0.033 mol/l sodium barbital-boric acid buffer on the cathode side at 250 V for 30 minutes. We also examined CK-MB sub-bands in myocardial extracts that showed only a broad MB2. When the extract was incubated with normal serum havings low CK activity at 37 degrees C at MB 1 sub-band appeared within a few hours. We examined the serial changes in these sub-bands in the serum of eight patients with acute myocardial infarction (AMI). The MB2 reached a peak 7-25 hours after onset of chest pain, and MB 1 reached a peak 0-12 hours later. The ratio of MB 2 to MB 1 was high at early phase of AMI. (about 7 hours after onset of chest pain, mean 1.7 S.D. 0.78) These results indicate that a high MB 2/MB 1 ratio is a more sensitive indicator of enzyme release from necrotic myocardium, and is useful for early diagnosis of AMI.