Lactate dehydrogenase-C4 (LDH) is a specific cell isoenzyme of spermatogenesis and spermatozoa of man and animals. Due to the lower levels of contaminating proteins, isolation of LDH C4 from spermatozoa had a lot of advantages. LDH C4 was isolated as follows: precipitation with ammonium sulfate, separation of LDH C4 from other LDH isoenzymes using immunosorbents, purification from contaminating proteins of oxamate-Sepharose. Antibodies to LDH1-3 immobilized on tresyl-Sepharose were used as immunosorbents. Elution of LDH C4 from oxamate-Sepharose by means of lithium lactate appeared inadequate therefore LDH C4 was eluted using 0.63 mM sodium pyruvate.