Glutathione peroxidase (GSHPx), (glutathione:H2O2 oxidoreductase, EC 1.11.1.9) was purified to homogeneity from human plasma. This resulted in a 6800-fold purification of the enzyme with a 2.8% yield. The purification process involved ammonium sulfate fractionation, DEAE-cellulose batch and column chromatographies, hydroxyapatite, and Sephadex G-200 and DEAE-Sephadex A-25 chromatographies. The major peak on DEAE-Sephadex A-25 column chromatography was found to be homogeneous on polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate (SDS). Relative mobility in nondenaturing polyacrylamide gel electrophoresis at pH 8.2 was 0.5 for the purified enzyme as detected by both protein staining and enzyme activity compared with 0.38 for erythrocyte GSHPx. The molecular weight of the plasma enzyme as determined by gel filtration was found to be approximately 100,000. SDS-gel electrophoresis of the plasma enzyme gave a subunit molecular weight of approximately 23,000. This suggests that the plasma enzyme exists as a tetramer in its native state, similar to that seen for the erythrocyte enzyme, but with slightly different mobility on SDS-gel electrophoresis. Plasma GSHPx, like the erythrocyte enzyme, was found to contain approximately four atoms of selenium per mole of protein. Utilizing iodinated concanavalin A, it was found that plasma GSHPx, but not the erythrocyte GSPx, is a glycoprotein. Purified plasma enzyme catalyzes both the reduction of tertiary butyl hydroperoxide and hydrogen peroxide. The apparent Km of plasma GSHPx for GSH is 5.3 mM and for tertiary butyl hydroperoxide it is 0.57 mM. Copper, mercury, and zinc strongly inhibit the enzyme activity of plasma GSHPx. Rabbit antibodies directed against the human erythrocyte GSHPx do not precipitate the enzyme activity of the purified plasma enzyme. Radioimmunoassay utilizing erythrocyte GSHPx and anti-erythrocyte GSHPx antibodies showed that less than 0.13% of the antigenically detectable protein is found in the purified GSHPx from plasma.