L-2-Oxothiazolidine-4-carboxylic acid (OTZ), a 5-oxoproline analog, is metabolized by 5-oxoprolinase and converted to cysteine, the rate-limiting amino acid for GSH synthesis, with the release of CO₂. [¹³C]OTZ (1.5 mg/kg) was used in 12 healthy men and women (ages 23-73 yr) to indirectly assess precursor mobilization for GSH synthesis when stores were reduced by 2 g acetaminophen. Expired breath samples were analyzed for ¹³CO₂, and results were analyzed using noncompartmental and two-compartment open minimal models. Results show an increase in ¹³C excretion (higher OTZ hydrolysis) when GSH stores were reduced and 5-oxoprolinase substrate utilization patterns, consequently, were altered (P < 0.01). A metabolic rate index (MRI) of the OTZ probe was found to be significantly higher after reduction of GSH content by acetaminophen (P < 0.05). The difference in adaptive capacity (difference between control and postacetaminophen metabolic rate indexes) was two times as large in the young than the old subjects (P < 0.01). These data support the use of [¹³C]OTZ as a probe to identify individuals who may be at risk for low GSH stores or who have an impaired capacity to synthesize GSH.