The cardiospecificity of the MB [myocardial] isoenzyme of creatine kinase, EC 2.7.3.2, (CK) with its associated diagnostic and research value has resulted in the development of a number of procedures for the separation and quantitation of the 3 isoenzymes of CK. Methods involving cellulose acetate electrophoresis, apposition incubation and fluoroscanning for quantitation are of limited linearity, insensitive, and irreproducible. A method was developed which overcomes these difficulties. This method is based on agarose gel electrophoresis, gel overlay incubation with optimization of all substrates and elution of the NADPH into solution for isoenzyme quantitation. This study has characterized this technique, used it to verify the extent to which the MB isoenzyme of CK is cardiospecific in rats and humans, studied its application to the diagnosis of acute myocardial infarction, and compared it with existing methods involving dithiothreitol activation and ion exchange chromatography.