The isolation and expression of a human liver cDNA encoding a 43 kDa protein with glycolate oxidase activity is described. The cDNA (HAO1) is 1128 bp and has a 1113 bp open reading frame. Northern blot analysis detects a 1.8 kb mRNA with expression restricted to the liver. The 370 amino acid protein has 89% sequence similarity to glycolate oxidase from mouse liver and 53% similarity to the spinach and Arabidopsis enzymes. The protein has glycolate oxidase activity in vitro, as measured by the reduction of 2, 6-dichlorophenolindophenol in the presence of flavin mononucleotide and glycolate. The genomic sequence is contained within eight exons and encompasses approximately 57 kb of chromosome 20p12. This paper offers the first full functional description of a gene encoding human glycolate oxidase.