The primary structure of human selenium-dependent phospholipid hydroperoxide glutathione peroxidase (GPX4) was determined by genomic cloning. The gene structure of GPX4 spans only 2.8 kb and consists of 7 exons. The coding sequence resides on all 7 exons, and the mitochondrial leader sequence is contained entirely within the first exon. The selenocysteine coding nucleotide resides on the third exon. The introns all commenced with the consensus nucleotide sequence GTR and ended with the consensus nucleotide sequence YAG. Analysis of the GPX4 gene sequence identified a potential alternative tissue-specific first exon. Chromosomal FISH studies placed the GPX4 gene at 19p13.3 location, and downstream of the 23 k-Da polypeptide DNA-directed RNA polymerase gene.