In the last 20 years, serum enzyme and isoenzyme levels have become the final arbiters by which acute myocardial infarction is diagnosed or excluded. We review the characteristics of these enzymes, the methods and limitations of commonly used assays, and data on diagnostic accuracy and clinical implications of enzyme levels in various settings and offer recommendations on their optimal use. Because of the poor sensitivity of single measurements of cardiac enzyme levels, these assays should not be used in the emergency room to exclude myocardial infarction. If myocardial infarction is suspected, levels of creatine kinase and its MB fraction should be measured at admission and about 12 and 24 hours later. If a myocardial infarction may have occurred more than 24 hours before evaluation, then lactate dehydrogenase isoenzyme measurements may increase diagnostic accuracy. Used properly, these assays are remarkably sensitive, but like all tests, optimal interpretation requires insight into technical pitfalls and other causes of misleading results.