Glycine undergoes decarboxylation in the glycine cleavage system (GCS) to yield CO(2), NH(3), and a 1-carbon unit. CO(2) also can be generated from the 2-carbon of glycine by 10-formyltetrahydrofolate-dehydrogenase and, after glycine-to-serine conversion by serine hydroxymethyltransferase, from the tricarboxylic acid cycle. To evaluate the relative fates of glycine carbons in CO(2) generation in healthy volunteers (3 male, 3 female, aged 21-26 y), primed, constant infusions were conducted using 9.26 micromol x h(-1) x kg(-1) of [1,2-(13)C]glycine and 1.87 micromol x h(-1) x kg(-1) of [5,5,5-(2)H(3)]leucine, followed by an infusion protocol using [1-(13)C]glycine as the glycine tracer. The time period between the infusion protocols was >6 mo. In vivo rates of whole-body glycine and leucine flux were nearly identical in protocols with [1,2-(13)C]glycine and [5,5,5-(2)H(3)]leucine and with [1-(13)C]glycine and [5,5,5-(2)H(3)]leucine tracers, which showed high reproducibility between the tracer protocols. Using the [1-(13)C]glycine tracer, breath CO(2) data showed a total rate of glycine decarboxylation of 96 +/- 8 micromol x h(-1) x kg(-1), which was 22 +/- 3% of whole-body glycine flux. In contrast, infusion of [1,2-(13)C]glycine yielded a glycine-to-CO(2) flux of 146 +/- 37 micromol x h(-1) x kg(-1) (P = 0.026). By difference, this implies a rate of CO(2) formation from the glycine 2-carbon of 51 +/- 40 micromol x h(-1) x kg(-1), which accounts for approximately 35% of the total CO(2) generated in glycine catabolism. These findings also indicate that approximately 65% of the CO(2) generation from glycine occurs by decarboxylation, primarily from the GCS. Further, these results suggest that the GCS is responsible for the entry of 5,10-methylenetetrahydrofolate into 1-carbon metabolism at a very high rate ( approximately 96 micromol x h(-1) x kg(-1)), which is approximately 20 times the demand for methyl groups for homocysteine remethylation.