A centrifugal analyzer method was developed for measuring the MB isoenzyme of creatine kinase (EC 2.7.3.2) in serum by use of a specific immunochemical technique that avoids interference from CK-BB and adenylate kinase. Enzymatic activity was measured kinetically at 30 degrees C with an optimized reagent containing creatine phosphate as substrate. The precision (CV) of the assay was 5 to 12 percent day-to-day (n = 39). The reference interval was 0 to 3.5 U per L (n = 45). Patient samples without detectable CK-MB in a widely-used electrophoretic assay contained up to 12 U per L of CK-MB by the new method. The new test was evaluated carefully in 99 patients consecutively admitted to the coronary care unit. Blood samples were obtained at frequent (four to eight hr) intervals. All patients with acute myocardial infarction (n = 27) had peak CK-MB greater than 7 U per L and greater than 3.5 percent of total CK. The predictive value of this result was 94 percent for the diagnosis of infarction. Abnormal results were documented at the same times (+/- four hr) following infarction by the electrophoretic and immunochemical techniques. Guidelines were evolved for interpreting the percent of MB (i.e., MB per total CK) at various time points. The test was reliable in documented early recurrent infarctions that occurred in three patients. The method appears to be an attractive alternative to electrophoretic techniques for use in diagnosis of acute myocardial infarction.