Estimation of infarct size from enzyme activities in plasma or serum presupposes known values of circulatory parameters such as the extravascular distribution volume Ve and the permeability constant P for the transport of enzyme between intravascular volume Vi and Ve. In man, parameter values are used that are extrapolated either from values found in the dog or from turnover studies of non-myocardial proteins. Large systematic errors can be introduced in this way, as demonstrated in this study. It is shown that by simultaneous determination of two different enzymes in the same patient, estimates of circulatory parameters are obtained. The method is applied to creatine kinase (CK) and alpha-hydroxybutyrate dehydrogenase (HBDH) plasma activities in 36 patients with acute myocardial infarction (AMI). The following results are obtained: 1. Exchange of enzyme between Vi and Ve is much slower and clearance of CK is much faster than presently assumed in the literature. 2. Release of CK and HBDH into the circulation is proportional to the amounts of these enzymes present in the myocardium. This finding is supported by data on early enzyme release. 3. Quantitation of HBDH release needs a two-compartment model, while for CK a one-compartment model can be used in good approximation. 4. Release of CK and HBDH after AMI continues up to 96 hours. 5. Using obtained parameter values, a simulated model demonstrates that estimation of clearance rates of CK from exponential fits on plasma levels results in large errors. This may explain recent conflicting results in validation of enzymatic estimates of infarct size.