Previous research has shown that glucose increases transamination of glutamine amino nitrogen with pyruvate. It is unclear whether glucose or glutamine provides the pyruvate used for transamination. In the current study, it was hypothesized that glucose provides pyruvate for transamination of glutamine amino nitrogen. This hypothesis was tested by tracing the metabolism of [2-(13)C]glucose in these cells incubated in the presence of [2-(13)C] glucose or [2-(13)C] glucose and glutamine using (13)C nuclear magnetic resonance. Glutamine supplementation increased alanine production but did not affect lactate production. The 1-(13)C, 2-(13)C, 3-(13)C, 1,2-(13)C, and 2,3-(13)C isotopomers of alanine and lactate were produced when glutamine was supplemented. Glutamine supplementation increased production of 2-(13)C, 1,2-(13)C, and 2,3-(13)C isotopomers of alanine but did not affect the production of isotopomers of lactate. The ratio of production of [2-(13)C]alanine to [3-(13)C]alanine was 37:1 when glutamine was present. The predominance of production of [2-(13)C]alanine vs. all other isotopomers demonstrates that a large proportion of the pyruvate used for transamination of glutamine amino nitrogen was derived from glycolysis.