Two overlapping clones were isolated from a human liver cDNA library in lambda gt11 that coded for human alkyl-dihydroxyacetonephosphate synthase using guinea pig and PCR-derived human cDNA probes. The open reading frame encodes a protein of 658 amino acids that shows a homology of 92% with the guinea pig homolog and a similarity of 98%. The peroxisomal targeting signal 2 that was recently identified in the presequence of the guinea pig enzyme appeared to be completely preserved in the human enzyme. Supportive confirmation for parts of the sequence of the mature protein was obtained from the Expressed Sequence Tags database of the National Center for Biotechnology Information. This database contained nine cDNA sequences, derived from seven independent clones, that correspond exactly to parts of the cDNA of human alkyl-dihydroxyacetonephosphate synthase. One of these clones most likely represents a not fully processed RNA with a putative intron containing an Alu sequence. An unexpected homology with D-lactate dehydrogenase (cytochrome C) precursor from Saccharomyces cerevisiae and with glycolate oxidase subunit D from Escherichia coli was also revealed.