A full-length cDNA clone encoding the major structural protein of trout CNS myelin 36K was isolated and sequenced. The deduced amino acid sequence did not reveal a putative transmembrane domain and exhibited no structural homology with any of the known myelin proteins. 36K instead shared characteristic structural elements with enzymes of the short-chain dehydrogenase family. The highest similarity in the database (60%), however, was obtained with a human protein of unknown function. By Northern blotting, a single mRNA species of about 2 kb was identified, which was expressed in brain tissue but not in liver. By in situ hybridization, a selective labeling of myelinating glial cells in the trout CNS but not in the PNS was revealed. The developmental appearance of the 36K transcript closely coincided with a period of active myelin deposition in most regions of the trout brain. As a first step in elucidating the structural and biochemical role of 36K for myelin formation and maintenance, we have overexpressed it in Escherichia coli as a soluble His-tag fusion protein and purified it in high yield by Ni+-chelated affinity chromatography. By SDS-PAGE, a single band of the expected molecular size was revealed, which heavily cross-reacted with polyclonal antibodies generated against the native protein. The results of circular dichroism spectroscopy are compatible with a betaalphabeta-barrel structure (Rossman fold), confirming the results of computer-assisted secondary structure predictions.