Among the proteins secreted by choroid plexus of vertebrates, one protein is much more abundant than all others. In mammals, birds, and reptiles this protein is transthyretin, a tetramer of identical 15-kDa subunits. In this study choroid plexus from frogs, tadpoles, and toads incubated in vitro were found to synthesize and secrete one predominant protein. However, this consisted of one single 20-kDa polypeptide chain. It was expressed throughout amphibian metamorphosis. Part of its amino acid sequence was determined and used for construction of oligonucleotides for polymerase chain reaction. The amplified DNA was used to screen a toad choroid plexus cDNA library. Full-length cDNA clones were isolated and sequenced. The derived amino acid sequence for the encoded protein was 183 amino acids long, including a 20-amino acid presegment. The calculated molecular weight of the mature protein was 18,500. Sequence comparison with other proteins showed that the protein belonged to the lipocalin superfamily. Its expression was highest in choroid plexus, much lower in other brain areas, and absent from liver. Since no transthyretin was detected in proteins secreted from amphibian choroid plexus, abundant synthesis and secretion of transthyretin in choroid plexus must have evolved only after the stage of the amphibians.