Vitamin D binding protein (DBP) was isolated from the plasma of the snapping turtle, Chelydra serpentina (mixed sex adult), and compared with the previously characterized dual function binding plasma protein that binds both thyroxine (T4) and vitamin D (tsTDBP) in an emydid turtle, Trachemys scripta. Purification of Chelydra serpentina DBP (csDBP) was accomplished by ion exchange chromatography, preparative polyacrylamide gel electrophoresis, and reverse-phase high-performance liquid chromatography. Estimates of increased purification (ca. 71-fold), recovery (ca. 1.5%), and corresponding plasma concentration (ca. 0.6 mg/ml) are confounded by interference with other proteins. A protein was identified that showed the same high affinity for 25-OH-cholecalciferol (vitamin D3) as the tsTDBP, and the two exhibited similar heat resistances in binding. The csDBP and tsTDBP had similar molecular weights by SDS-PAGE (ca. 58 kDa), showed immunological cross reactivity, and differed by only three residues, representing conservative substitutions, in the 30 NH2-terminal amino acid sequence. A slightly lesser homology (up to 89% similarity based on conservative substitutions) was seen with three mammalian DBPs. However, unlike tsTDBP, the DBP from the snapper did not bind T4. These data support the view that the DBP of emydid turtles secondarily evolved a second T4 binding site, and this is probably independent of the D3 binding domain.