The affinity of purified human vitamin D-binding protein from serum (DBP) for 25-hydroxyvitamin D3 (25-OHD3) and 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] was measured in the presence of free fatty acids (FFA), cholesterol, prostaglandins and several drugs. Mono- and polyunsaturated fatty acids markedly decreased the affinity of both 25-OHD3 and 1,25-(OH)2D3 for DBP, whereas saturated fatty acids (stearic and arachidic acid), cholesterol, cholesterol esters, retinol, retinoic acid and prostaglandins (A1 and E1) did not affect the apparent affinity. Several chemicals known to decrease the binding of thyroxine to its plasma-binding protein did not affect the affinity of DBP. The apparent affinity of DBP for both 25-OHD3 and 1,25-(OH)2D3 decreased 2.4- to 4.6-fold in the presence of 36 microM of linoleic or arachidonic acid, respectively. Only a molar ratio of FFA:DBP higher than 10,000 was able to decrease the binding of 25-OHD3 to DBP by 20%. Much smaller ratio's of FFA:DBP (25 for arachidonic and 45 for oleic acid), however, decreased the binding of 1,25-(OH)2D3 to DBP. These latter ratio's are well within the physiological range. The addition of human albumin in a physiological albumin:DBP molar ratio did not impair the inhibitory effect of linoleic acid on the binding of [3H]25-OHD3 to DBP. The binding and bioavailability of vitamin D metabolites thus might be altered by mono- and polyunsaturated but not by saturated fatty acids.