To measure the source and rate of mucosal glutathione (GSH) synthesis, fed piglets (28 days old; 7.7 kg) received a 6-h infusion of intragastric [U-13C]glutamate (n = 11) either with (n = 5) or without (n = 6) an intragastric infusion of [1-13C]glycine (0-6 h) and [1,2-13C2(U-13C)]glycine (3-6 h). Eighty-four percent of the labeled mucosal GSH-glutamate and 86% of the luminal GSH-glutamate was 13C5. The tracer-to-tracee ratio of GSH-[U-13C]glutamate was 75% of that of mucosal glutamate. Sixty percent of the labeled mucosal glutamate was 13C1, 13C2, or 13C3, but the tracer-to-tracee ratios of these isotopomers in GSH-glutamate were not significantly different from zero. After 3 h of infusion, the tracer-to-tracee ratio of GSH-[U-13C]glycine was 46%, and after 6 h of infusion GSH-[13C1]glycine was 82% of that of mucosal glycine. This suggested that the half-life of mucosal GSH was 2.7 +/- 0.1 h. We concluded that, in fed piglets, mucosal GSH-glutamate derived largely from the direct metabolism of enteral glutamate rather than from glutamate that was metabolized within the mucosa.