Specific serum binding of 25-hydroxy-cholecalciferol (25-OHD3) was measured by saturation analysis in rats of various ages, and during vitamin D deprivation. The serum binding capacity for 25-OHD3 was observed to increase until age 6-8 weeks, then decline and remain stable thereafter at 3.7 x 10(-6) M. The 25-hydroxyvitamin D (25-OHD) concentration decreased in serum from rats fed vitamin D-free diet (t1/2 = 7 days). Rats fed 2 IU of vitamin D3/g of diet maintained stable serum levels of 25-OHD at 10-12 ng/ml. Serum binding capacity and affinity for 25-OHD3 was not affected by vitamin D deprivation or hypocalcemia. In addition, the binding affinity did not differ as a function of age (Kd = 3.3 x 10(-9) M). Since normal serum concentrations of 25-OHD in the rat are 2-5 x 10(-8) M, only 1-2% of the serum binding sites for this sterol are occupied under physiological conditions.