The minimum requirements for four essential trace metals were examined in both thermophilic and mesophilic methane fermentation of glucose. The minimum requirement here was defined as the amount that supports the effluent soluble chemical oxygen demand (COD) of 1000 mg/ L or less for the duration of several times the hydraulic retention time (HRT). For this purpose, completely mixed reactors were run semi-continuously at 5 days HRT, with 10 g/L substrate COD. The minimum requirements determined as milligrams per gram COD removed were 0.45, 0.049, 0.054, and 0.24 in the thermophilic system and 0.20, 0.0063, 0.017, and 0.049 in the mesophilic system for iron, nickel, cobalt, and zinc, respectively. The thermophilic system required larger amounts (2.2 to 7.8 times) of the trace metals than the mesophilic system, indicating greater assimilation by biomass and/or less bioavailability under thermophilic conditions. The results can give quantitative information on trace metal nutrition for successful methane fermentation.