Mouse lactate dehydrogenase-B cDNAs were isolated from cDNA libraries of macrophage (ICR strain) and thymus (F1 hybrid of C57BL/6 and CBA strains), and their nucleotide sequences determined. The lactate dehydrogenase-B cDNA insert of thymus clone mB188 consists of the protein-coding sequence (1002 nucleotides), the 5' (46 nucleotides) and 3' (190 nucleotides) non-coding regions, and poly(A) tail (19 nucleotides), while macrophage clone mB168 contains a partial lactate dehydrogenase cDNA insert from codon no. 55 to the poly(A) tail. Seven silent nucleotide substitutions at codon no. 142, 143, 186, 187, 241, 285 and 292, as well as a single nucleotide change in the 3' non-coding region, were found between these different strains of mice. The predicted sequence of 333 amino acids, excluding initiation methionine, was confirmed by sequencing and/or compositional analyses of a total of 103 (31%) amino acids from tryptic peptides of mouse lactate dehydrogenase-B protein. The nucleotide sequence of the mouse coding region for lactate dehydrogenase B shows 86% identity with that of the human isoenzyme, and only eight of the 139 nucleotide differences resulted in amino acid substitutions at residues 10, 13, 14, 17, 52, 132, 236 and 317. The rates of nucleotide substitutions at synonymous and nonsynonymous sites in the mammalian lactate dehydrogenase genes are calculated. The rates of synonymous substitutions for lactate dehydrogenase genes A (muscle) and B (heart) are considerably higher than the average rate computed from human and rodent genes. The rates of nonsynonymous substitutions for lactate dehydrogenase genes A (muscle) and B (heart), particularly the latter, are highly conservative. The rates of synonymous and nonsynonymous substitutions for the lactate dehydrogenase-C gene are about the same as the average rates for mammalian genes. A phylogenetic tree of vertebrate lactate dehydrogenase protein sequences is constructed. In agreement with the previous results, this analysis further indicates that lactate dehydrogenase-C gene branched off earlier than did lactate dehydrogenase-A and lactate dehydrogenase-B genes.