Technical Abstract: The objective of this study was to evaluate oxidative metabolism of glucose, glutamate, and glutamine by isolated ovine enterocytes in presence of other metabolic fuels in vitro. Mucosal enterocytes were isolated from crossbred wether sheep (n=6) fed a mixed forage-concentrate diet, and incubated for 90 min with 1 mM U-14C-glucose, -glutamate, or -glutamine and alternative substrates (AS; water as negative control, acetate, propionate, butyrate, glucose, glutamate, or glutamine) at concentrations of 0.1, 1.0, and 10.0 mM. Oxidation of labeled substrates to CO2 and net production of lactate and pyruvate in incubation media were measured. Oxidation of glucose and glutamine to CO2 was decreased by all AS except acetate, and the extent of reduction differed by type and concentration of AS in incubation media. Our observations that glutamine oxidation can be reduced by presence of AS is contrary to observations in the literature using enterocytes from non-ruminants, indicating that ruminant enterocytes might rely on glutamine to a less extent as an energy source. Total glucose utilization was reduced by propionate (10 mM) by 16% compared to control, but was not affected by other AS. Glutamate oxidation to CO2 was reduced by the presence of propionate (10 mM) or glutamine (1.0 and 10 mM), but not by that of the other AS. Acetate did not affect oxidation of glucose, glutamate, and glutamine. Propionate reduced oxidation of glucose and glutamate only at the highest concentration (10 mM), indicating that the sparing effects of propionate on substrate oxidation is affected its concentration in the incubation media. These observations indicate that ruminant DME can alter substrate preference for oxidative metabolism depending on type and concentration of available AS.