The pyridoxal-5'-phosphate-dependent enzymes (B-6 enzymes), that operate in the metabolism of amino acids, are of multiple evolutionary origin. To estimate their rates of evolution, a total of 180 sequences of 21 B-6 enzymes from distantly related eukaryotic species were compared. The enzymes belong to all four evolutionarily independent families of B-6 enzymes with different folds, i.e., the large a family, the beta family, the D-alanine aminotransferase family, and the alanine racemase family. Their unit evolutionary periods, i.e., the time for a 1% sequence difference to accumulate between branches, ranged from 4.6 to 45.1 million years. Both, fastest changing serine pyruvate aminotransferase and most slowly changing glutamate decarboxylase are members of the a family. The evolutionary rates of the few enzymes belonging to the other three families were interspersed among those of the a family members. Enzymes that catalyze the same reaction, e.g., transamination or amino acid decarboxylation, with different substrates show widely varying rates. The absence of correlations of the rate of evolution with either protein fold or type of catalyzed reaction suggests that individual functional constraints have determined the differential rates of evolution of B-6 enzymes.