The objective of these experiments was to determine the fate of tritium from the 5 position of proline and to assess the validity of its loss to H2O as a measure of proline oxidation. When [5-3H]proline was fed to barley (Hordeum vulgare) leaves, tritium was recovered in H2O and metabolites such as glutamate, glutamine, organic acids, aspartate, asparagine, and γ-aminobutyrate. Collectively these metabolites, which are oxidation products of proline, accounted for 8% of the 3H recovered after 5 hours. In spite of the amount recovered in metabolites, the rates of proline oxidation estimated by measuring 3H2O recovery from [5-3H]proline were only slightly lower than rates estimated by incorporation of 14C into oxidized products and loss of 14C from total proline. Therefore, 3H2O recovery from [5-3H]proline is useful in assessing the effects of stress on proline metabolism.