Human tumor cell lines cultured in 75Se-containing media demonstrate four major 75Se-labeled cellular proteins (57, 22, 18, and 12 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Among these selenoproteins, an enzymatic activity is known only for the 22-kDa protein, since this protein has been identified as the monomer of glutathione peroxidase. However, all tested cell lines also contained a peroxidase activity with phospholipid hydroperoxides that is completely accounted for by the other selenoenzyme, phospholipid hydroperoxide glutathione peroxidase (PHGPX) (Ursini, F., Maiorino, M., and Gregolin, C. (1985) Biochim. Biophys. Acta 839, 62-70). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of 75Se-labeled proteins separated by gel permeation chromatography supported the identification of PHGPX as the monomeric protein matching the 18 kDa band. This paper is the first report on the identification of PHGPX in human cells.