We describe the rapid profiling of isoenzymes by use of microparticulate anion-exchange chromatography supports and a continuous, post-separation enzyme detector in a high-performance liquid chromatograph. Chromatographic analysis and enzyme detection are fully automated and provide excellent reproducibility. Factors affecting the isoenzyme profile and detector response characteristics are assessed. Lactate dehydrogenase and creatine kinase isoenzymes in tissue extracts, control materials used as electrophoretic standards, and serum were profiled by this method to establish the resolution and reliability of the method. We show the clinical use of this method in detecting changes in these isoenzymes in serum associated with acute myocardial infarction.