Lactate fermentation occurs in anaerobic organisms or in aerobic cells that are undergoing very high rates of glycolysis. In these cells, NADH generated in glycolysis cannot be reoxidized to NAD⁺ . When this situation happens, NADH is oxidized to NAD⁺ by reducing pyruvate to lactate. The enzyme catalyzing this reaction is lactate dehydrogenase. The equilibrium for this reaction lies far in favor of formation of lactate.

Until recently it was thought that lactate accumulation in skeletal muscle was largely a consequence of anaerobic metabolism, which occurs when the need for tissues to generate energy exceeds their capacity to oxidize the pyruvate produced in glycolysis. Recent metabolic studies, including ³¹P NMR analyses of the levels of phosphorylated intermediates in living muscle cells during exercise, suggest that lactate is actually an intermediate and not a metabolic "dead end," whose only fate is reconversion to pyruvate. These studies show that even in fully oxygenated muscle tissue, as much as 50% of the glucose metabolized is converted to lactate. This may represent a means for coordinating energy-storing and energy-generating pathways in different tissues, but the mechanisms involved are not yet clear.

Lactate dehydrogenase was the first enzyme that established the structural basis for the existence of isoenzymes (different forms of an enzyme resulting from variations in amino acid sequence). Most tissues contain five isoenzymes of lactate dehydrogenase that can be resolved electrophoretically.