Methanosphaera stadtmanae (DSM 3091) is a methanogen that requires H-2 and CH3OH for methanogenesis. The organism does not possess an F-420-dependent hydrogenase and only low levels of F-420. It does however possess NADP(+):F-420 oxidoreductase activity. The NADP(+):F-420 oxidoreductase, the enzyme which catalyses the electron transfer between NADP(+) and F-420 in this organism, was purified and characterized. NAD(+), NADH, FMN, and FAD could not be used as electron acceptors. Optimal pH for F-420 reduction was 6.0, and 8.5 for NADP(+) reduction. During the purification process, it was noted that precipitation with (NH4)(2)SO4 increased total activity 16-fold but reduced the stability of the enzyme. However, recombination of cell-free extracts with resuspended 65-90% (NH4)(2)SO4 pellet returned activity to near cell-free extract levels. Neither high salt or protease inhibitors were effective in stabilizing the activity of the partially purified enzyme. The purified enzyme from M. stadtmanae possessed a molecular weight of 148 kDa as determined by gel filtration chromatography and native-PAGE, consisting of alpha, beta, and gamma subunits of 60, 50, and 45 kDa, respectively, using SDS-PAGE. The K-m values were 370 muM for NADP(+), 142 muM for NADPH, 62.5 muM for F-420, and 7.7 muM for F420H2. These values were different from the K-m values observed in the cell-free extract.