Through the screening of microorganisms capable of utilizing α-methylserine, three representative strains belonging to the bacterial genera Paracoccus, Aminobacter, and Ensifer were selected as potent producers of α-methylserine hydroxymethyltransferase, an enzyme that catalyzes the interconversion between α-methyl-l-serine and d-alanine via tetrahydrofolate. Among these strains, Paracoccus sp. AJ110402 was selected as the strain exhibiting the highest α-methylserine hydroxymethyltransferase activity. The enzyme was purified to homogeneity from a cell-free extract of this strain. The native enzyme is a homodimer with apparent molecular mass of 85kDa and contains 1mol of pyridoxal-5′-phosphate per mol of the subunit. The Km for α-methyl-l-serine and tetrahydrofolate was 0.54mM and 73μM, respectively. The gene from Paracoccus sp. AJ110402 encoding α-methylserine hydroxymethyltransferase was cloned and expressed in Escherichia coli. Sequence analysis revealed an open reading frame of 1278bp, encoding a polypeptide with a calculated molecular mass of 46.0kDa. Using E. coli cells as whole-cell catalysts, 9.7mmol of α-methyl-l-serine was stereoselectively obtained from 15mmol of d-alanine and 13.2mmol of formaldehyde.