Tobacco (Nicotiana tabacum var. Havana Seed) leaf discs were allowed to photosynthesize for 3 to 20 minutes in the presence of (14)CO(2) and (3)H(2)O. Several metabolites of the Calvin cycle and photorespiratory pathway were isolated and purified and the (3)H:(14)C values measured. Glycolate had a 5- to 10-fold higher (3)H:(14)C than the Calvin cycle intermediate 3-phosphoglyceric acid, or its end product sucrose. The glycolate oxidase inhibitor alpha-hydroxy-2-pyridinemethanesulfonic acid caused glycolate to accumulate in the tissue and lowered the (3)H:(14)C in glycolate to a value similar to that in 3-phosphoglyceric acid. Phosphoglycolate, a possible precursor of glycolate arising from the Calvin cycle, exhibited a (3)H:(14)C value similar to 3-phosphoglyceric acid under all conditions. The finding of a (3)H enrichment in glycolate suggests that another source of glycolate, possibly the reduction of glyoxylate, exists in leaf tissue. Analyses of incorporation of (3)H into the pro-2R and pro-2S hydrogens of glycolate, in the presence and absence of alpha-hydroxy-2-pyridinemethanesulfonic acid, suggest an alternative source of glycolate. Biochemical mechanisms to account for (3)H enrichment into glycolate are evaluated.