Thyroxine (T4) binding proteins in turtle plasma were compared with those of various mammalian and nonmammalian (fish, birds) species using gel filtration chromatography (Bio-Gel). High-affinity T4 binding was observed in all mammals studied, in the chicken, and in one species of turtle, Trachemys scripta. This high affinity T4 binding protein (TBP) appears to be the major component of T4 transport in the turtle; selective removal of this TBP greatly diminished binding activity in T. scripta plasma. In all other nonmammalian species studied (including other turtles), T4 binding appeared to be associated primarily with relatively low affinity components such as albumin. Albumin concentrations in turtle (T. scripta, and Chelydra serpentina) plasmas were examined by measurement of dry weight, protein assay (Bradford), radioimmunoassay, and electrophoretic densitometry. While C. serpentina may have total protein concentrations approaching that of human plasma, albumin content in both turtles was estimated at about 3-4 mg/ml, less than 10% that of human plasma albumin; larger molecular weight components that do not bind T4 comprise a greater proportion of turtle plasma protein. Additionally, purified T. scripta albumin exhibits lower T4 binding than does human albumin; binding to turtle albumin is also reduced by fatty acids. Taken together, these results indicate a lesser role for albumin in plasma T4 binding in T. scripta than in humans, and, thus, a larger role for the T4 binding protein, TBP. Other turtles lacking a TBP may rely wholly on low affinity albumin binding that also has a lower capacity than that in mammals.