Orthologous homologs of lactate dehydrogenase-A (LDH-A) (EC 188.8.131.52; NAD+:lactate oxidoreductase) of six barracuda species (genus Sphyraena) display differences in Michaelis-Menten constants (apparent Km) for substrate (pyruvate) and cofactor (NADH) that reflect evolution at different habitat temperatures. Significant increases in Km with increasing measurement temperature occur for all homologs, yet Km at normal body temperatures is similar among species because of the inverse relationship between adaptation temperature and Km. Thermal stabilities of the homologs also differ. To determine the amino acid substitutions responsible for differences in Km and thermal stability, peptide mapping of the LDH-As of all six species was first performed. Then, the amino acid sequences of the three homologs having the most similar peptide maps, those of the north temperate species, S. argentea, the subtropical species, S. lucasana, and the south temperate species, S. idiastes, were deduced from the respective cDNA sequences. At most, there were four amino acid substitutions between any pair of species, none of which occurred in the loop or substrate binding sites of the enzymes. The sequence of LDH-A from S. lucasana differs from that of S. idiastes only at position 8. The homolog of S. argentea differs from the other two sequences at positions 8, 61, 68, and 223. We used a full-length cDNA clone of LDH-A of S. lucasana to test, by site-directed mutagenesis, the importance of these sequence changes in establishing the observed differences in kinetics and thermal stability. Differences in sequence at sites 61 and/or 68 appear to account for the differences in Km between the LDH-As of S. argentea and S. lucasana. Differences at position 8 appear to account for the difference in thermal stability between the homologs of S. argentea and S. lucasana. Evolutionary adaptation of proteins to temperature thus may be achieved by minor changes in sequence at locations outside of active sites, and these changes may independently affect kinetic properties and thermal stabilities.