The distribution of aspartate racemase (EC 5.1.1.13) in various kinds of bacteria demonstrated that the enzyme occurs in lactic acid bacteria, such as Streptococcus species and Lactobacillus species. The enzyme from Streptococcus thermophilus IAM10064 was more thermostable than that from Streptococcus lactis IAM1198 which contained the enzyme most abundantly among the lactic acid bacteria we examined here. We purified the enzyme about 3400-fold to homogeneity from cell-free extract of S. thermophilus, which is composed of two identical subunits with a molecular weight of 28,000 as a homodimer. The enzyme utilizes specifically aspartate as a substrate, but not alanine and glutamate. Maximal reaction velocity was observed at 37 degrees C and around pH 8.0. The sequence of the NH2-terminal amino acids of the enzyme was determined to be Met-Glu-Asn-Phe-Phe-Ser-Ile-Leu-Gly-XXX-Met-Gly-Thr-Met-Ala-Thr-Glu-Ser- Phe-.