sn-Glycerol-l-phosphate (G-1-P) dehydrogenase is the key enzyme for biosynthesis of the enantiomeric glycerophosphate backbone of ether phospholipids of Archaea. The gene encoding this enzyme in Methanobacterium thermoautotrophicum (egsA) was used to construct an expression plasmid pTrcG1Pdh for Escherichia coli. The G-1-P dehydrogenase activity of E. coli XL1-blue/pTrcG1Pdh was maximal 8-10 h after induction. The expressed G-1-P dehydrogenase was purified 4300-fold from the soluble fraction to homogeniety after 4300 times purification by a procedure consisting of fractionation with ammonium sulfate precipitation, and hydrophobic and ion-exchange chromatographies. The yield was about 70%. The V-max value for the forward reaction to produce G-1-P was 740 units (mu mol/min)/mg, with a K-m of 0.21 mM for NADH and 0.39 mM for dihydroxyacetone phosphate. The K-m＇s for G-I-P and NAD(+) in the backward reaction were 10.5 and 0.46 mM, respectively. These kinetic constants are similar to those for the enzyme from M. thermoautotrophicum. G-I-P dehydrogenase was successfully used to analyze the stereospecificity of glycerophosphate, which is an intermediate of phospholipid biosynthesis and glycerol metabolism; the rate of NADH formation was proportional to the G-1-P concentration up to 3 mM in the presence of 0.02 unit of the purified enzyme.