A variety of sulfur-containing compounds were investigated for use as medium reductant and sulfur sources for growth of the acclimated-mixed methanogens. In Hungate tube cultures, the optimum concentrations of sulfur compounds for growth and CH4 formation were sulfide, 0.9 mmol; cysteine, 3.6 mmol; thiosulfate, 0.675 mmol; elemental sultur (S degrees), 0.45 gl(-1); glutathione, 2.5 mmol; SO42-, 3.6 mmol; SO32-, 0.9 mmol; and S2O52- 0.09 gl(-1). In comparison with sulfide as sole sulfur source, CH4 formation was about the same while higher increments of cell density were obtained by using cysteine, thiosulfate, S degrees and glutathione. Other sulfur-containing compounds tested resulted in decreased CH4 formation but with a higher increment of cell density. When contaminating sulfidogenic bacteria were inhibited by addition of antibiotics to the medium which are known to affect eubacteria but nor methanogens, only a slight amount of hydrogen sulfide was produced by assimilatory reduction of methanogens, and the growth of the methanogens was poor, especially in the case of growing on SO42-, SO32-, thiosulfate or glutathione, indicating that evolved hydrogen sulfide was mainly due to a sulfide-producer that existed symbiotically with predominant methanogens in the cultures. However, in the case of cysteine or S degrees as sulfur sources, in spite of inhibition of contaminating bacteria, sufficient sulfide was reduced by methanogens to support growth and methane formation effectively. These sulfur compounds were also tested for their ability to enhance and restore growth and CH4 production with sulfur-depleted cells in culture tubes. The results of the present study show that, for the growth of the acclimated-mixed methanogens, volatile and malodorous sulfide can be replaced with the sulfur-containing compounds described above. Copyright (C) 1996 Elsevier Science Ltd.