Lactate dehydrogenase (LDH) is an enzyme of special interest due to its key position in anaerobic metabolic pathways. Work on the structure and developmental expression of LDH led to formulation of the isoenzyme concept as well as elucidation of the biological significance of isoenzymes. Tissue isoenzyme compositional changes are mediated by changes in pattern of gene expression for two primary LDH gene loci in vertebrates. Developmental shifts in LDH gene expression prompted other experimental interventions to alter gene expression schedule and enzyme synthesis in order to pinpoint underlying genetic and molecular control mechanisms. LDH isoenzymes in tissues can be separated by electrophoresis and then quantitated by scanning densitometry; however, these methods require specialized instruments. Enzyme specific activity can be assessed spectrophotometrically, but enzymatic activity determination alone does not provide quantitation of LDH isoenzyme(s). The present work was conducted to establish a spectrophotometric enzyme assay procedure based on differential substrate inhibition to quantitate tissue LDH isoenzymes. The procedure was then used to assess developmentally related alterations in LDH isoenzymes in cardiac tissue of newborn and young adult rats. Results show that heart tissue in 5-day old newborn rats possesses approximately equal proportions of muscle type (anaerobic) and cardiac type (aerobic) LDH isoforms. As normal development transpires, heart tissue LDH isoenzyme proportion shifts with substantial decrease in anaerobic form which is accompanied by marked augmentation in aerobic form.