The enzyme which seems to be responsible for the formation of the enantiomeric configuration of the glycerophosphate backbone (sn-glycerol-1-phosphate) of archaeal ether lipids was purified from a methanogenic archaeon, Methanobacterium thermoautotrophicum, and characterized. The enzyme, sn-glycerol-1-phosphate: NAD(P)+ oxidoreductase (sn-glycerol-1-phosphate dehydrogenase), was purified 7,600-fold from a cell free extract by ammonium sulfate fractionation and seven steps of chromatography. The final preparation exhibited a specific activity of 617 micromol/min/mg (Vmax) and gave a single band corresponding to 38 kDa on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The native enzyme showed an apparent molecular mass of 302 kDa on gel-filtration chromatography, indicating it is present as a homooctamer. Maximum activity was observed at 75 degrees C at near neutral pH. The activity was stimulated by potassium ions. The Km for dihydroxyacetone phosphate was 7.5 times smaller than that for sn-glycerol-1-phosphate, suggesting that the formation of sn-glycerol-1-phosphate is the natural direction in the cell. Under the assay conditions used, no product inhibition was observed. The N-terminal amino acid sequence was determined.