The preferential utilization of different electron donors and their effects on the nitrate reduction and methanogenesis in a mixed, mesophilic (35°C) methanogenic culture were investigated. Batch methanogenic cultures were fed with dextrin/peptone (D/P), propionate, acetate, and H2/CO2 at an initial COD of 500 mg/L and an initial nitrate concentration of 50 mg N/L. Immediate cessation of methane production was observed in all nitrate-amended cultures. Methane production completely recovered in the D/P- and acetate-fed cultures, and partially recovered or did not recover in the propionate- and H2/CO2-fed, nitrate-amended cultures, respectively. Accumulation of denitrification intermediates was observed in both the propionate- and H2/CO2-fed cultures, which resulted in inhibition of fermentation and/or methanogenesis. The fastest and the slowest nitrate reduction were observed in the acetate- and propionate-fed cultures, respectively.