Résumé : Human and rat erythrocytes were found to generate 3HOH from D-[6(N)- 3H]glucose. The rate of 3HOH production represented 7-10% of the glycolytic flux. The generation of 3HOH appeared attributable, in part at least, to the detritiation of [3-3H]pyruvate during the interconversion of the 2-keto acid and L-alanine in the reaction catalyzed by glutamate-pyruvate transaminase. Indeed, purified pig heart glutamate-pyruvate transaminase, as well as homogenates prepared from rat erythrocytes or pancreatic islets, catalyzed the generation of 3HOH from L-[3-3H)alanine. When the production of tritiated pyruvate from L-[3-3H]alanine was coupled to the conversion of the 2-keto acid to L-lactate, the production of 3HOH accounted for one-third of the reaction velocity, the latter failing to display isotopic discrimination. In these experiments, the production of 3HOH was abolished by aminooxyacetate. Likewise, in intact rat erythrocytes, aminooxyacetate inhibited the generation of 3HOH and tritiated L-alanine from D-[6- 3H]glucose (or D-[1-3H]glucose), as well as the generation of 3HOH from L- [3-3H]alanine. In pancreatic islets, however, aminooxyacetate failed to affect significantly the generation of 3HOH from D-[6-3H]glucose. These findings indicate that the generation of 3HOH from D-[6-3H]glucose is mainly attributable to an intermolecular tritium transfer in transaminase reaction, at least in cells devoid of mitochondria.