Two experiments were conducted to determine the effect of trace mineral source on sulfate metabolism in cattle, and a third trial explored the effect of sulfur source on thiamin disappearance in vitro. In the first experiment, crossbred heifers (335.54 kg) were supplemented with an organically complexed source of Cu, Zn, Mn, and Co; an inorganic source of Cu, Zn, Mn, and Co; or no added trace minerals. Heifers were assigned to one of two water treatments, no added sulfate (NS); (39,5 mg/kg sulfate) or high-sulfate (WS); (1,810 mg/kg sulfate). Average daily gain for heifers consuming NS water was higher (P = 0,04) than for those consuming WS water for the first 28d. Water source had no effect on gain for 29 to 56 (P = 0.07) or 0 to 56d (P = 0.77). Feed-to-gain was lower (P = 0,01) for NS heifers than for WS treatments for the first 28d. Water source had no effect on feed-to-gain for 29 to 56 (P = 0.06) or 0 to 56d (P = 0.44). Mineral source had no effect (P = 0.18) on cattle performance. In Experiment 2, an in vitro incubation study was used to determine the effect of organically complexed, sulfate, and oxide mineral sources on dry matter disappearance and culture pH when ruminal fluid donors were consuming water with no added sulfate (NS) or 2,000 mg/kg sulfate (WS). A level x source interaction (P = 0.01) was found for in vitro dry matter disappearance (IVDMD). Differences in IVDMD were found within ground com (P = 0.01) and com starch (P = 0.04) substrate groups when donors were consuming NS water. With the addition of sulfate to donor drinking water, IVDMD differences (P - 0.003) were only found within treatments with ground corn as a substrate. Differences (P - 0.02) in culture pH were noted when com starch was used as a substrate and donors were consuming NS water. With the addition of sulfate to donor drinking water, differences were noted for culture pH when substrates included ground corn (P - 0.03) and alfalfa (P = 0.02). In Experiment 3, an in vitro study was conducted to determine whether sulfur affected thiamin disappearance. Sulfur was introduced to tubes containing ground com and ruminal fluid. Sulfur was added as sulfate, sulfide, sulfite, or L-cysteine. Tubes treated with sulfate had higher (P < 0.01) thiamin concentrations than tubes tested with cysteine at 0 h. Sulfide had higher (P = 0.04) thiamin concentrations than cysteine at 0 h. No differences in thiamin concentration were found for sulfrir source at 24 (P = 0,06) or 48 h (P = 0.35). At 0 h, tubes with 0.0, 0.5, and 1.0 mg S/mL ruminal fluid had lower (P < 0.01) thiamin concentrations than treatments with 0,25 mg/mL. At 24 h, 0.25 mg/mL treatments had lower (P < 0.01) thiamin concentrations than the other three treatments. At 48 h, thiamin concentrations were higher (P = 0.02) for 0.0 mg/mL than 0.25 and 0.5 mg/mL, lower (P < 0.01) than 1.0 mg/mL treatments. Tubes treated with 0.25 and 0.5 mg/mL did not differ (P = 0.11), but had lower (P < 0.01) thiamin concentrations than 1.0 mg/mL treatments.