A new fluorescent compound, factor(420) (F(420)), which is involved in the hydrogen metabolism of hydrogen-grown Methanobacterium strain M.o.H. has been isolated and purified. Acid hydrolysis of this compound with 6 m HCl for 24 hr releases a ninhydrin-positive compound (glutamic acid), an acid-stable chromophore, phosphate, and an ether-soluble phenolic component. Factor(420) may be reduced by either sodium dithionite or sodium borohydride at pH 7.3 with concomitant loss of its fluorescence and its major absorption peak at 420 nm. Crude cell-free extracts of strain M.o.H. reduce F(420) only under a hydrogen atmosphere. F(420) is photolabile aerobically in neutral and basic solutions, whereas the acid-stable chromophore is not photolabile under these conditions. An approximate molecular weight of 630 +/- 8% for F(420) was determined by Sephadex G-25 chromatography. At the present time, F(420) is proposed as a trivial name for the unknown fluorescent compound because of its strong absorption maximum of 420 nm at pH 7.