A cDNA which codes for human cytosolic serine hydroxymethyltransferase (Garrow et al., 1993, J. Biol. Chem. 268, 11910-11916) has been cloned into a pT7-7 vector as a NdeI-EcoRI insert. HMS174 (de3) cells were transformed with this plasmid and, after induction with isopropyl thiogalactoside, expressed a catalytically active serine hydroxymethyltransferase. The enzyme was purified and shown to be the expressed human enzyme by N-terminal amino acid sequencing. About 225 mg of pure enzyme can be obtained from a 20-liter culture. Spectral characteristics of the bound pyridoxal phosphate were essentially identical to the spectral properties of rabbit cytosolic serine hydroxymethyltransferase. Kinetic constants for the natural substrates L-serine and tetrahydrofolate were also similar to the values obtained previously for the rabbit cytosolic enzyme.