The lactate dehydrogenase (EC 1.1.1.27, LDH) inhibitors were isolated from the LDH-free crude mitochondrial fraction of skeletal muscle in Syrian hamster (Mesocricetus auratus) and Korean native cattle (Bos taurus coreanae). The LDH inhibitor in skeletal muscle of M. auratus was successfully isolated by the treatment with 175 mM NaCl and ultrasonic. The LDH inhibitor in skeletal muscle of B. taurus coreanae was highly stable to heat and LDH fu isozyme was largely inhibited by the LDH inhibitor. The molecular weight of inhibitor was 22 kDa. Inhibitor played an important role in the binding of LDH with the mitochondria in tissues of skeletal muscle, kidney and liver except heart.