Variations in plasma sulfur amino acid (SAA) pools are associated with disease risks, but little information is available about the factors affecting plasma SAA pools. Drug metabolism by glutathione (GSH) and sulfate conjugation can, in principle, represent a quantitatively important burden on SAA supply. The present study was designed to determine whether therapeutic doses of acetaminophen (APAP) alter SAA metabolism in healthy human adults. A double-blind, crossover design incorporating four treatment periods with diets providing 100% of the recommended dietary allowance (RDA) for SAA without or with APAP (15 mg/kg) and 0% RDA for SAA without or with APAP, in randomized order. After a 3-day equilibration period, chemically defined diets with 100 or 0% RDA for SAA were given for 2 complete days. On day 3, APAP or placebo was given in two successive doses (6-h interval), and timed plasma samples were collected. With SAA intake at 100% RDA, APAP administration oxidized the plasma cysteine/cystine redox potential (E(h)CySS) but not the plasma GSH/GSSG redox potential (E(h)GSSG). The extent of oxidation caused by APAP was similar to that seen with 0% SAA and no APAP. However, APAP administration with 0% SAA did not cause further oxidation beyond APAP or 0% SAA alone. In contrast, an oxidation of the plasma E(h)GSSG was apparent for SAA insufficiency only with APAP. The results suggest a need to evaluate possible effects of APAP in association with SAA insufficiency as a contributing factor in disease risk.