A method is presented for the preparation and assay of lactate dehydrogenase isoenzymes 1 and 2 as pure fractions. The total method involves the use of heat treatment to destroy the heat-labile fractions (isoenzymes 3, 4 and 5); fractionation using an ion-exchange resin; and immunochemical blocking of the muscle subunit, to remove any contamination of the fraction containing isoenzyme 1 with isoenzyme 2 and vice versa. Finally, the enzyme activities in the various fractions are measured using a standard kinetic assay procedure. Estimates can be obtained of all five isoenzymes if the heat treatment step is omitted.