Changes in the proportions of individual isoforms of the MM isoenzyme of creatinine kinase (CK; EC 22.214.171.124) in plasma promptly reflect both myocardial infarction and coronary recanalization. However, quantitative methods developed thus far are too slow or cumbersome for routine use in making clinical decisions. We report a convenient, quantitative chromatofocusing assay with on-line fluorometric detection of isoform activity in the column eluent that provides results within 40 min from the time of sample application. Sample eluted from a microbore chromatofocusing column (1.8-mL bed volume) is split between a reaction stream, into which CK reagents are added, and a reference stream. After incubation at 37°C, NADPH formed by reaction of isoforms with CK reagent is detected at 340 nm. The system can detect activity of individual isoforms in plasma samples having total CK activity ≥ 21 U/L (30°C). Results correlated closely with those obtained by previously validated, but slow, chromatofocusing (r = 0.98, n = 30) and protein immunoblotting (r = 0.90, n = 20) procedures.