The GPX1, GPX2 and GPX3 genes of Saccharomyces cerevisiae have been reported previously to encode glutathione peroxidases (GPxs). We re-examined the sequence alignments of these proteins with GPxs from higher eukaryotes. Sequence identities, particularly with phospholipid hydroperoxide glutathione peroxidases (PHGPxs), were enhanced markedly by introduction to the yeast sequences of gaps that are characteristic of PHGPxs. PHGPx-like activity was detectable in extracts from wild type S. cerevisiae and was diminished in extracts from gpx1D, gpx2D and gpx3D deletion mutants; PHGPx activity was almost absent in a gpx1D/gpx2D/gpx3D triple mutant. Studies with cloned GPX1, GPX2 and GPX3 expressed heterologously in Escherichia coli confirmed that these genes encode proteins with PHGPx activity. An S. cerevisiae gpx1D/gpx2D/gpx3D mutant was defective for growth in medium supplemented with the oxidation-sensitive polyunsaturated fatty acid linolenate (18:3). This phenotype was more marked than sensitivity to H2O2. Unlike H2O2 toxicity, delayed toxicity of 18:3 towards gpx1D/gpx2D/gpx3D cells was correlated with the gradual incorporation of 18:3 into S. cerevisiae membrane lipids and was suppressible with a-tocopherol, an inhibitor of lipid peroxidation. The results show that the GPX genes of S. cerevisiae previously reported to encode GPxs encode PHGPxs, and that these enzymes protect yeast against phospholipid hydroperoxides as well as non-phospholipid peroxides. This is the first report of an organism that expresses PHGPx from more than one gene, and that produces PHGPx in the absence of a GPx.