The presence of transthyretin in mammals and birds, but not amphibia, suggested that transthyretin expression first appeared in stem reptiles. Therefore, transthyretin synthesis was studied in a lizard. Transthyretin synthesis in choroid plexus pieces from Tiliqua rugosa was demonstrated by incorporation of radiactive amino acids. Oligonucleotides corresponding to conserved regions of transthyretin were used as primers in polymerase chain reaction with lizard choroid plexus cDNA. Amplified DNA was used to screen a lizard choroid plexus cDNA library. A full-length transthyretin cDNA clone was isolated and sequenced. A three-dimensional model of lizard transthyretin was obtained by homology modeling. The central channel of transthyretin, containing the thyroxine-binding site, was found to be completely conserved between reptiles and mammals. Transthyretin expression was not detected in lizard liver. These data suggest that transthyretin first evolved in the choroid plexus of the brain. Due to a change in tissue distribution of gene expression, occurring much later during evolution, transthyretin also became a plasma protein, synthesized in the liver.