At least two gene duplication events have led to the three lactate dehydrogenase (LDH; EC 1.1.1.27) isozymes (LDH-A, LDH-B, and LDH-C) of chordates. The prevailing model for the evolution of the LDH loci involves duplication of a primordial LDH locus near the origin of vertebrates, giving rise to Ldh-A and LU-B. A third locus, designated Ldh-C, is expressed in the spermatocytes of mammals and a single family of birds and in the eye or liver tissues of teleost fishes. Ldh-C might have arisen independently in these taxa as duplications of either Ldh-A or Ldh-B. Several authors have challenged this traditional hypothesis on the basis of amino acid sequence and immunological similarity of the three LDH isozymes. They suggest that the primordial LDH gene was duplicated to form Lk-C and a locus that later gave rise to Ldh-A and Lk-B. We have differentiated between these hypotheses by determining the cDNA sequence of the retinaspecific LDH-C from a teleost, Fundulus heteroclitus. On the basis of amino acid sequence similarity, we conclude that the LDH-C isozymes in fish and mammals are not orthologous but derive from independent gene duplications. Furthermore, our phylogenetic analyses support previous hypotheses that teleost LUh-C is derived from a duplication of the LU-B locus.Many workers have emphasized the importance of gene duplication as a mechanism of evolution and biological diversification (e.g., refs.