Positive effects on fiber degradation have been observed when supplemental cobalt (Co) was added to diets for ruminants, but dose-dependent effects of different Co sources on ruminal fermentation have not been tested. Our objective was to determine the effects of different sources and concentrations of Co on in vitro fermentation rate, fermentation end products, and DM and NDF disappearance. Ruminal fluid was collected from heifers fed a wheat straw based diet (49%) with no supplemental Co, and fermentation substrate (46% NFC, 25% NDF, 22% CP) contained no measurable Co. Different inclusion levels (0.0, 0.1, 0.5, 1.0, 2.0, 5.0, 10.0, and 15.0 ppm) of Co glucoheptonate (S1) and Co carbonate (S2) were tested in vitro during study 1. Gas production was recorded every 15 min, and after 24 h, pH was measured and contents of each flask were used to determine NDF and DM disappearance (NDFD and DMD). Experiment 2 evaluated the effects of Co (S1 and S2 at 0, 0.33, 1, 3, and 9 ppm) on gas production, ruminal VFA and NH₃ concentration. In both studies, each treatment combination had 4 replicates and samples were incubated for 24 h; asymptotic gas production curves were modeled with NLIN of SAS using the Gauss-Newton fit method. Gas production kinetic values and all other data were modeled to assess the effects of Co concentration, source, and their interaction. Differences were declared at P < 0.05 and tendencies at P < 0.10. NDFD was increased by S1 at concentrations between 0.1 and 1.0 ppm (55.0 ± 1.8%), but decreased at 15 ppm (30.9%). S2 had no effect on NDFD across the concentrations tested. Moderate levels of Co tended to increase DMD, and S1 significantly increased this parameter compared with S2 (63.5 vs. 61.1% ± 1.6%), but interactions were not detected. Effects of Co source on gas production kinetics and pH change were inconsistent between experiments, largely because of dramatic negative effects at 15 ppm. Propionate concentration increased by S1 at 1.0 ppm, but not by S2 (dose by source interaction). Doses of 9 ppm Co decreased branched-chain VFA and NH₃ concentrations after 24-h incubation. In summary, Co does affect ruminal fermentation; however, concentrations of 9 ppm and greater may be inhibitory, particularly with a highly soluble source of Co.