We studied the effects of sulfur and 9,10-anthraquinone on in vitro ruminal fermentation and production of hydrogen sulfide. A complete, pelleted diet containing 26.8% acid detergent fiber, 15.9% crude protein, and 0.25 to 0.29% sulfur was used as the basal substrate. Fermentations were conducted using the basal substrate and various sulfur additions (elemental sulfur, thiosulfate, calcium sulfate, and sodium sulfate) with or without varying amounts of 9,10-anthraquinone. An increase in the sulfur content of the substrate to > 1.0% with various sources of sulfur had minimal effects on concentrations of volatile fatty acids, but the production of hydrogen sulfide increased. High amounts of 9,10-anthraquinone (10 and 25 ppm of fluid) decreased the molar proportion of acetate and decreased the production of methane and hydrogen sulfide. However, 9,10-anthraquinone increased the molar proportions of propionate and butyrate. Approximately 70% of 9,10-anthraquinone was recovered after 24 h of in vitro ruminal fermentation. These findings suggest that 9,10-anthraquinone has the potential to reduce the production of methane and hydrogen sulfide in ruminal fermentations.