L-5-oxoproline (L-5-OP) is an intermediate in glutathione synthesis, possibly limited by cysteine availability. Urinary 5-OP excretion has been proposed as a measure of glycine availability. We investigated whether 5 days of dietary sulfur amino acid (SAA-free) or glycine (Gly-free) restriction affects plasma kinetics of 5-OP and urinary excretion of L- and D-5-OP in 6 healthy men. On day 6, L-5-[1-¹³C]oxoproline and [3,3-²H₂]cysteine were infused intravenously for 8 h (3 h fast/5 h fed). In a control study (adequate amino acid mixture), plasma oxoproline fluxes were 37.8 ± 13.8 (SD) and 38.4 ± 14.8 µmol · kg¹ · h¹; oxidation accounted for 85% of flux. Cysteine flux was 47.9 ± 8.5 and 43.2 ± 8.5 µmol · kg¹ · h¹ for fast and fed phases, respectively. Urinary excretion of L- and D-5-OP was 70 ± 34 and 31.1 ± 13.3 µmol/mmol creatinine, respectively, during days 3-5, and 46.4 ± 13.9 and 22.4 ± 8.3 µmol/mmol over the 8-h tracer study. The 5-OP flux for the Gly-free diet was higher (P = 0.018) and tended to be higher for the SAA-free diet (P = 0.057) when compared with the control diet. Oxidation rates were higher on the Gly-free (P = 0.005) and SAA-free (P = 0.03) diets. Cysteine fluxes were lower on the the Gly-free (P = 0.01) and the SAA-free diets (P = 0.001) compared with the control diet. Rates of L-5-OP excretion were unchanged by withdrawal of SAA or Gly for 5 days but increased on day 6 (P = 0.005 and P = 0.019, respectively). Thus acute changes in the dietary availability of SAA and Gly alter oxoproline kinetics and urinary 5-OP excretion.