The metaxin genes of zebrafish have been investigated by determining the sequences of metaxin cDNAs and analyzing the translated amino acid sequences. A zebrafish cDNA corresponding to a third metaxin gene was identified. Zebrafish cDNAs representing metaxins 1 and 2, previously described in human and mouse, were also identified. The zebrafish metaxin genes are designated mtx1, mtx2 and mtx3, following zebrafish nomenclature guidelines. The zebrafish metaxin 3 (ZMTX3) cDNA codes for a protein of 313 amino acids (MW 35,208), while the ZMTX1 and ZMTX2 cDNAs specify proteins of 317 residues (MW 35,906) and 274 residues (MW 30,852), respectively. Alignment of the ZMTX3 and ZMTX1 amino acid sequences revealed 40% identities, while 26% identities were found for the ZMTX3/ZMTX2 alignment. A phylogenetic tree showed that the metaxins share a common ancestry, with the grouping of the zebrafish sequences with the homologous human and mouse sequences. Analysis of the domain structure of the zebrafish metaxins uncovered a glutathione S-transferase (GST) domain for each protein and, in addition, a thioredoxin-like domain for ZMTX2. A region of transmembrane helices was found near the C-terminus for the ZMTX1 protein. In addition, regions of alpha helix were seen to be the predominant feature of zebrafish metaxin secondary structure, particularly for ZMTX2 and ZMTX3. The ZMTX3 cDNA sequence has the greatest homology to a human sequence at cytogenetic location 5q14.1, close to the thrombospondin 4 gene (THBS4). Also, the mouse metaxin 3 homologue is adjacent to Thbs4 at 13C3.