We have demonstrated that lactate dehydrogenase is not solely a cytosolic enzyme by the isolation and purification of the enzyme from the mitochondria of human liver. Treatment of the mitochondria with digitonin reveals the LD activity to be associated with the inner membrane-inner matrix and the outer membrane. The mitochondrial LD consists of two fractions separated by ion exchange and affinity chromatography. The first mitochondrial fraction, LD-Mt1, with isoelectric points of 9.8, 9.6, and 4.8, has subunit components of 14500 and 34000 daltons. The second mitochondrial fraction, LD-Mt2, is similar to cytosolic LD-5 with respect to both isoelectric points and subunit molecular weight. The first mitochondrial fraction, LD-Mt1, has physical characteristics previously associated with the isoenzyme LD-6.