Chromium(VI) reductase activity was measured in ultra-filtrates of rat liver and kidney after various pretreatments in vitro at 37°C and pH 7.0. Preincubation of ultrafiltrates with L-ascorbate oxidase (EC 1.10.3.3), which specifically eliminated ascorbate, blocked ˜80% of the Cr(VI) reductase activity. Heat-denatured ascorbate oxidase had no effect on Cr(VI) reductase activity in ultrafiltrates. Preincubation of ultrafiltrates with N-ethylmaleimide, which non-specifically blocked sulfhydryls, including reduced glutathione, decreased Cr(VI) reductase activity by only 20%. Treatment of male Sprague-Dawley rats with phorone decreased non-protein sulfhydryl (NPSH) levels in rat liver by >90% and tripled reduced ascorbate levels 2 h after treatment. Ultrafiltrates of liver prepared from phorone-treated rats had twice the Cr(VI) reductase activity of control ultrafiltrates, and >95% of this activity could be blocked by preincubation with ascorbate oxidase. Treatment of rats with sodium dichromate (20 mg/kg) caused a significant decrease in ascorbate levels in kidney but not liver, and no change in NPSH levels in kidney or liver, 15 min after treatment. We conclude that ascorbate is the major reductant of Cr(VI) in rat liver and kidney ultrafiltrates and may well be the major non-enzymatic reductant of Cr(VI) in rat liver and kidney in vivo.