Retinol transport and metabolism have been well characterized in mammals, however very little is known in fish. To study the mechanism by which fish RBP is able to remain in plasma besides its small molecular size, we isolated RBP cDNA from a carp liver cDNA library. Comparison of the deduced amino acid sequence with that of known vertebrate RBPs showed that carp RBP has high homology to the other cloned vertebrate RBPs, but it lacks the C-terminal tetrapeptide, RNL(S)L, which is most likely involved in the interaction with TTR in mammalian RBPs. In addition, the primary structure of carp RBP contains two consensus N-linked glycosylation sites that represent a unique feature. We have obtained experimental evidence, by in vitro and in vivo expression experiments, that both sites are indeed glycosylated. We have also characterized the protein as a complex type N-linked glycoprotein by lectin binding assay, neuraminidase and Endo H and F digestion. Inhibition of glycosylation by tunicamycin treatment of transfected cells caused a great reduction of RBP secretion. Since kidney filtration of anionic proteins is less than half that of neutral protein of the same size, this finding strongly suggests that the amount of carp RBP filtration through kidney glomeruli may be reduced by a glycosylation dependent increase in the molecular size and negative charge of the protein. A second unique feature of carp RBP as secretory protein is the presence of a non conserved N-terminal hydrophobic domain that functions as an insertion signal, but is not cleaved co-translationally and remains in the secreted RBP.