The relation between seasonal changes in plasma thyroxine (T,) and T, binding activity was studied in three species of turtle previously shown to differ in the nature of their binding proteins; additional direct measurements of a high affinity T, binding protein (TBP) were made in one species using RIA. In the painted turtle, Chrysemyspicta (Family Emydidae), a species with large seasonal changes in plasma T,, the binding protein measured by activity and RIA showed significant seasonality. Binding activity correlated with direct estimates of TBP by RIA. TBP was minimal upon emergence from hibernation and maximal in late summer (July-August), about a month after the peak in plasma T,. In contrast, two other nonemydid species, the green turtle (Chelonia mydas) and Mexican tortoise (Gopherus flauomarginatus) showed relatively low plasma T, and binding activity with no significant seasonality in either. Electrophoretic studies (PAGE) confirmed that T, binding in C. picta is almost wholly associated with a high affinity, low capacity TBP; whereas, it is confined t o high capacity sites (probably albumin) in the other species. The transport of T, is clearly handled differently in these three turtles. In the green turtle and tortoise, one would expect relatively constant free T, (fT4) throughout the year. Marked seasonal fluctuations (an order of magnitude) in the ratios of TBP to total T, in C. picta due to the temporal lag between the two may cause profound changes in fT, with peaks in late spring.