5,10-Methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum strain delta H was purified to homogeneity with nearly complete recovery. The aerobically stable monofunctional enzyme catalyzed the reversible oxidation of 5,10-methylene-5,6,7,8-tetrahydromethanopterin to its 5,10-methenyl derivative. For the reaction a midpoint potential E'0 = - 362 mV was calculated at 60 degrees C. The methanogenic electron carrier coenzyme F420 was strictly required as the co-substrate. The dehydrogenase (Mr 216,000) was purified as an apparent hexamer of six identical 36 kDa subunits. Oxidation of 5,10-methylenetetrahydromethanopterin coupled to coenzyme F420 reduction catalyzed by the dehydrogenase with a turnover number of 2400 S-1 proceeded via a ternary complex mechanism. High concentrations of monovalent cations markedly stimulated the reaction.