In order to study functions of selenoprotein P in human plasma, its level was lowered via two techniques, chromatography on Sepharose-bound heparin, or immunoprecipitation; Western blot analysis showed that both techniques were effective at substantially lowering selenoprotein P levels in plasma. When peroxynitrite was infused to maintain a 0.9 microM steady-state concentration, plasma made deficient in selenoprotein P diminished benzoate hydroxylation significantly less than control plasma. Similar differences were found for protein 3-nitrotyrosine formation, determined by Western blot analysis. Conversely, in a selenoprotein P-enriched plasma preparation obtained via heparin-Sepharose chromatography, protection against benzoate hydroxylation was above controls. Likewise, a supernatant from control plasma that had been exposed to anti-selenoprotein P antibodies was less efficient in preventing oxidation and nitration reactions of peroxynitrite than the supernatant from plasma exposed to a non-specific antibody (rabbit anti-sheep IgG). These data demonstrate a role of selenoprotein P in human plasma in the defense against peroxynitrite.