Glutathione-dependent formaldehyde dehydrogenase (FDH; EC 1.2.1.1) has been purified 3900-fold from maize cell-suspension cultures to a specific activity of 4.68 mumol (mg protein)-1 min-1. The homogeneous enzyme consisted of two identical subunits with a molecular mass of 42 kDa, and an isoelectric point of 5.8. Eight tryptic peptides were sequenced and gave a perfect fit to the protein sequence derived from maize Fdh cDNA (J. Fliegmann and H. Sandermann, 1997, Plant Mol Biol 34: 843-854). There was 62% identity with the eucaryotic FDH consensus sequence. Michaelis constants of approx. 20 microns (formaldehyde), approx. 50 microns (glutathione) and approx. 31 microns (NAD+) were determined for the maize enzyme as well as for FDH partially purified from dog lung. Besides S-hydroxymethylglutathione, pentanol-1, octanol-1, and omega-hydroxy-fatty acids served as substrates for both FDH preparations. The unusual substrate specificity indicates that FDH may be involved in the detoxification of long-chain lipid peroxidation products.