sn-Glycerol-1-phosphate dehydrogenase is responsible for the formation of the sn-glycerol-1-phosphate backbone of archaeal lipids. [4-3H]NADH that had 3H at the R side was produced from [4-3H]NAD and glucose with glucose dehydrogenase (a pro-S type enzyme). The 3H of this [4-3H]NADH was transferred to dihydroxyacetonephosphate during the sn-glycerol-1-phosphate dehydrogenase reaction. On the contrary, in a similar reaction using alcohol dehydrogenase (a pro-R type enzyme), 3H was not incorporated into glycerophosphate. These results confirmed a prediction of the tertiary structure of sn-glycerol-1-phosphate dehydrogenase by homology modeling.