The lactate dehydrogenase (EC 1.1.1.27, LDH) isozymes in tissues from Acanthogobius hasta were characterized by biochemical, immunochemical and kinetic methods. The activities of LDH in skeletal muscle and eye tissues were 65.30 and 53.25 units, but LDH activities in heart and liver tissues were very low. LDH/CS (EC 4.1.3.7, citrate synthase) in skeletal muscle was the highest as 22.29. Specific activities of LDH in brain, eye and skeletal muscle were 56.45, 38.04 and 11.0 units/mg, respectively. The LDH isozymes in tissues were separated by polyacrylamide gel electrophoresis after immunoprecipitation with antiserum against A4, B4, eye-specific C4 and liver-specific C4. LDH A4 isozymes were detected predominantly in skeletal muscle, brain and eye tissues, and B4 isozyme was detected in heart. Anodal eye-specific C4 and cathodal liver-specific C4 were coexpressed in A. hasta. The eye-specific C4 isozyme showed higher activity in eye tissue, but liver-specific C4 isozyme showed lower activity in liver. As a result, one part of molecular structures in A4 and C4, A4 and B4, and eye-specific C4 and liver-specific C4 were similar, but in B4 and C4 were different with each other. Therefore the subunit A may be conservative in evolution, and the evolution of subunit B seems to be faster than that of subunit A. The LDH A4 isozyme of skeletal muscle was purified in the fraction from elution with NAD+ containing buffer of affinity chromatography and eye-specific C4 isozyme was eluted right after A4, so the structure of eye-specific C4 isozyme is similar to A4. And LDH activity remained 35.22-43.47% as a result of the inhibition by pyruvate, the Michaelis-Menten constant values for pyruvate was 0.080-0.098 mM, and Vmax were 153.85 units, 35.09 units in skeletal muscle and eye, respectively. Also the B4 isozyme was the thermo-stablest and C4 was stabler than A4 isozyme. The optimum pH of LDH was 6.5. The results mentioned above indicate that isozymes in tissues showed the properties between LDH A4 and B4 isozyme as A. hasta was adapted to hypoxic conditions. Also LDH seems to function more effectively under anaerobic condition because LDH in skeletal muscle and eye tissues have high affinity for pyruvate.