Thyroid function was examined during the annual cycle of Richardson's ground squirrel, Spermophilus richardsoni. A number of facets were measured to facilitate comparison of thyroid function in active, dormant, and aroused animals. This report discusses changes in the serum thyroid hormone and binding as assessed by radioimmunoassay, equilibrium dialysis, and competitive binding assays. During the hibernation phase (both dormant and aroused), total serum T3 (trioodo-L-thyronine) and T4 (L-thyroxine) are elevated over active levels, two- to fivefold and four- to sixfold, respectively. However, in dormant squirrels, both free T3 and free T4 are reduced compared with both active and aroused phases of the annual cycle, while in aroused squirrels there is an increase in free T3 but no change in free T4 compared with active squirrels. The difference between changes in total and free thyroid hormone levels in the three groups is due to changes in serum binding of thyroid hormone. There is a more than twofold increase in the capacity of a saturable T3-binding site in serum of both dormant and aroused squirrels, and there is an increase in serum binding affinity at the low core temperature of dormant squirrels (6 degrees). Therefore, even though serum total T3 and T4 are elevated during dormancy, free T3 and T4 levels are reduced to half of the levels in active squirrels as a consequence of increased serum binding capacity and affinity. In aroused animals, however, increased serum binding capacity only partially buffers the increase in total T3 and T4, so that free thyroid hormone levels exceed those of active squirrels.