Abstract The objective of this study was to evaluate the effects of different sources of cobalt in a lactating dairy cow diet on in vitro ruminal fermentation. All diets provided the same amount of supplemental Co (1 mg per kg of diet DM) included in a premix with a targeted inclusion level of 2.5% of diet DM. Treatments were cobalt carbonate (CON), and three alternative Co sources; alternative source 1 (AS1), alternative source 2 (AS2), and alternative source 3 (AS3). Eight dual-flow continuous culture fermenters were used in a replicated 4 × 4 Latin square. Experimental periods were 10 d (7 d adaptation and 3 d sample collection). Composite samples of daily effluents were collected and analyzed for NH3-N, lactate, and VFA concentration as well as for nutrient digestibility. Samples of ruminal content were collected from each fermenter at 0, 1, 2, 4, 6, and 8 h after feeding to determine lactate, NH3-N, and pH kinetics. All data were analyzed using PROC MIXED of SAS and the repeated variable of time was included for pH, lactate, and NH3-N. There were neither treatment effects nor interactions between treatment and time for pH, NH3-N, and lactate concentrations, indicating a similar Co kinetics across treatments. With regards to lactate pool, we did not observe treatment effects for L-lactate; however, we observed treatment effects for D-lactate and total-lactate as seen in Table 1. Analyses of total and individual VFA, N utilization, and nutrient degradation are still undergoing, hence, it is possible that Co sources have effects on ruminal fermentation.