CDP-2,3-di-O-geranylgeranyl-sn-glycerol:L-serine O-archaetidyltransferase (archaetidylserine synthase) activity in cell extracts of Methanothermobacter thermautotrophicus cells was characterized. The enzyme catalyzed the formation of unsaturated archaetidylserine from CDP-unsaturated archaeol and L-serine. The identity of the reaction products was confirmed by thin-layer chromatography, fast atom bombardment-mass spectrum analysis, and chemical degradation. The enzyme showed maximal activity in the presence of 10 mM Mn2+ and 1% Triton X-100. Among various synthetic substrate analogs, both enantiomers of CDP-unsaturated archaeols with ether-linked geranylgeranyl chains and CDP-saturated archaeol with ether-linked phytanyl chains were similarly active toward the archaetidylserine synthase. The activity on the ester analog of the substrate was two to three times higher than that on the corresponding ether-type substrate. The activity of D-serine with the enzyme was 30% of that observed for L-serine. A trace amount of an acid-labile, unsaturated archaetidylserine intermediate was detected in the cells by a pulse-labeling experiment. A gene (MT1027) in M. thermautotrophicus genome annotated as the gene encoding phosphatidylserine synthase was found to be homologous to Bacillus subtilis pssA but not to Escherichia coli pssA. The substrate specificity of phosphatidylserine synthase from B. subtilis was quite similar to that observed for the M. thermautotrophicus archaetidylserine synthase, while the E. coli enzyme had a strong preference for CDP-1,2-diacyl-sn-glycerol. It was concluded that M. thermautotrophicus archaetidylserine synthase belongs to subclass II phosphatidylserine synthase (B. subtilis type) on the basis of not only homology but also substrate specificity and some enzymatic properties. The possibility that a gene encoding the subclass II phosphatidylserine synthase might be transferred from a bacterium to an ancestor of methanogens is discussed.