The sequence and structure of human testis-specific L-lactate dehydrogenase [LDHC4, LDHX; (L)-lactate: NAD+ oxidoreductase, EC 1.1.1.27] has been derived from analysis of a complementary DNA (cDNA) clone comprising the complete protein coding region of the enzyme. From the deduced amino acid sequence, human LDHC4 is as different from rodent LDHC4 (73% homology) as it is from human LDHA4 (76% homology) and porcine LDHB4 (68% homology). Subunit homologies are consistent with the conclusion that the LDHC gene arose by at least two independent duplication events. Furthermore, the lower degree of homology between mouse and human LDHC4 and the appearance of this isozyme late in evolution suggests a higher rate of mutation in the mammalian LDHC genes than in the LDHA and -B genes. Comparison of exposed amino acid residues of discrete antigenic determinants of mouse and human LDHC4 reveals significant differences. Knowledge of the human LDHC4 sequence will help design human-specific peptides useful in the development of a contraceptive vaccine.