Coenzyme F,,,-dependent methylenetetrahydromethanopterin dehydrogenase from methanogenic Archaea catalyzes the reversible transfer of a hydride ion from C14a of N5,Mo-methylenetetrahydromethanopterin to C5 of coenzyme F,,,. In this study, we report that this hydride transfer proceeds stereospecifically from the Re face at C14a to the Si face at C5. The results were obtained by using chirally 3H-labelled N5,Mo-methylenetetrahydromethanopterin generated via Re-face-specific H,-forming N5,Wo-methylenetetrahydromethanopterin dehydrogenase and by analyzing reduced coenzyme F,,, via Si-face-specific F,,,-reducing hydrogenase.Keywords. Coenzyme F,,, ; stereospecificity ; methylenetetrahydromethanopterin dehydrogenase ; hydrogenase.Coenzyme F4,,-dependent methylene tetrahydromethanopterin dehydrogenase catalyzes the reversible conversion of N5,Mo-methylenetetrahydromethanopterin (CH,=H,MPT) and coenzyme F,,, (F,,,) to W,Mo-methenyl-H,MPT (CH= H4MPT+) and reduced coenzyme F,,, (F4zoH,) (reaction a; for structures of CH,=H,MPT, CH=KMPT+, F,,, and F42, HZ see The enzyme is found in methanogenic Archaea (Schworer and Thauer, 1991) and Archaeoglobus species (Moller-Zinkhan et al., 1989;Vorholt et al., 1994 on lactate plus sulfate, the enzyme catalyzes the oxidation of CH,=H,MPT to CH-H,MPT+ as an intermediary step in CO, formation (Keltjens and Vogels, 1993; Thauer and Kunow, 1995).