Glutamine carbon entry into the tricarboxylic acid (TCA) cycle was assessed in small intestinal epithelial cells by measuring CO₂ production from [1-¹⁴C]glutamine, and these data together with [U-¹⁴C]glutamine data were used to calculate fractional oxidation rates for glutamine. CO₂ production from either [1-¹⁴C]glutamine or [U-¹⁴C]glutamine showed saturation kinetics, and the concentration needed to achieve the half-maximal rate of CO₂ production was 0.7 and 0.4 mmol/l, respectively. Maximal rate for [1-¹⁴C]glutamine was twice that for [U-¹⁴C]glutamine. Increasing glutamine concentration did not cause proportional increases in glutamine entry into the TCA cycle and glutamine oxidation. Consequently, fractional oxidation of glutamine decreased with increasing glutamine concentration. Fractional oxidation could be predicted from the rate at which glutamine carbon entered the TCA cycle. (Aminooxy)acetic acid, an aminotransferase inhibitor, reduced entry of glutamine into the TCA cycle and increased fractional oxidation of glutamine. Glutamate carbon entered the TCA cycle at about one-half the rate of glutamine-derived glutamate carbon and had a higher fractional oxidation rate when provided at equivalent concentrations to glutamine. These differences in the rate of entry predictably account for the differences in the metabolic fate of glutamine vs. glutamate carbon.