Effects of monensin (MON) and S on in vitro fermentation and H(2)S production were evaluated in 2 experiments. In Exp. 1, 2 ruminally cannulated steers were adapted (>14 d) to a 75% concentrate diet [steam-flaked corn (SFC)-based], and ruminal fluid was collected approximately 4 h after feeding. Substrate composed (DM basis) of 85.2% SFC, 9% alfalfa hay, 5% cottonseed meal, and 0.8% urea was added with ruminal fluid and buffer to sealed 125-mL serum bottles to allow for gas collection. A Na(2)SO(4) solution was added to yield S equivalent to 0.2, 0.4, and 0.8% of substrate DM, and MON was included at 0, 2, 4, and 6 mg/L of culture volume. Bottle head-space gas was analyzed for H(2)S. No MON (P = 0.29) or MON x S interaction (P = 0.41) effects were detected for H(2)S production. Increasing S linearly increased (P < 0.01) H(2)S production (micromoles/g of fermented DM). The IVDMD (average 70.0%) was not affected by MON (P = 0.93), S (P = 0.18), or the MON x S interaction (P = 0.56). Total VFA concentrations were not affected by MON (P = 0.87), S (P = 0.14), or the MON x S interaction (P = 0.86), but increasing MON linearly decreased (P <or= 0.01) molar proportions of acetate, butyrate, and the acetate:propionate ratio (A:P), and linearly increased (P < 0.01) the proportion of propionate. In Exp. 2, 2 additional ruminally cannulated Jersey steers were adapted (>21 d) to a 75% concentrate diet (SFC base) that contained 15% (DM basis) wet corn distillers grains plus solubles (WDGS) and MON at 22 mg/kg of DM. In vitro substrate DM was composed of 75.4% SFC, 15% WDGS, 9% alfalfa hay, and 0.6% urea, and S and MON concentrations were the same as in Exp. 1. No effects of MON (P = 0.93) or the MON x S interaction (P = 0.99) were noted for H(2)S production; however, increasing S linearly increased (P < 0.01) H(2)S production. No effects of MON (P = 0.16), S (P = 0.43), or the MON x S interaction (P = 0.10) were noted for IVDMD (average 70.9%). Total VFA concentrations were not affected by MON (P = 0.40), S (P = 0.26), or the MON x S interaction (P = 0.59). As in Exp. 1, increasing MON linearly decreased (P < 0.05) molar proportions of acetate, butyrate, and A:P and linearly increased (P < 0.01) propionate. Increasing S concentration increased in vitro H(2)S production, but S did not affect VFA concentrations and proportions. Monensin did not affect in vitro H(2)S production, and changes in VFA molar proportions were evident with MON regardless of S concentration.