Mechanistic studies have been undertaken on the coenzyme F420 dependent formate dehydrogenase from Methanobacterium formicicum. The enzyme was specific for the si face hydride transfer to C5 of F420 and joins three other F420-recognizing methanogen enzymes in this stereospecificity, consistent perhaps with a common type of binding site for this 8-hydroxy-5-deazariboflavin. While catalysis probably occurs by hydride transfer from formate to the enzyme to generate an EH2 species and then by hydride transfer back out to F420, the formate-derived hydrogen exchanged with solvent protons before transfer back out to F420. The kinetics of hydride transfer from formate revealed that this step is not rate determining, which suggests that the rate-determining step is an internal electron transfer. The deflavo formate dehydrogenase was amenable to reconstitution with flavin analogues. The enzyme was sensitive to alterations in FAD structure in the 6-, 7-, and 8-loci of the benzenoid moiety in the isoalloxazine ring.