Greater than 99% of the total circulating 1,25-dihydroxyvitamin D [1,25-(OH)2D] is bound to proteins such as the vitamin D-binding protein (DBP) and albumin; in the normal human only 0.4% of the circulating 1,25-(OH)2D is free. Although it is often assumed that only the free concentration of 1,25-(OH)2D is available to cells, this has not been demonstrated. In particular, it is not clear whether the DBPs facilitate 1,25-(OH)2D entry into target cells or serve only to transport these metabolites within the circulation. To address this question, we evaluated one of the best characterized target tissue responses to 1,25-(OH)2D, namely its ability to inhibit its own production and induce that of 24,25-(OH)2D, using one of the most sensitive cells, the human foreskin keratinocyte. We incubated keratinocytes in the presence of 1,25-(OH)2D (from 10(-11)-10(-8) M) in medium containing albumin (from 0.1-10%) or serum (from 0.1-10%) for 4 h [to inhibit the 25-hydroxyvitamin D (250HD) 1 alpha-hydroxylase] or 16 h (to induce the 250HD 24-hydroxylase) before evaluating [3H]250HD metabolism by these cells during a 1-h incubation in serum- and albumin-free medium. The free fraction of 1,25-(OH)2D was determined in each medium by centrifugal ultrafiltration and varied from 36% to 0.57% in direct proportion to the albumin or serum in the medium. Increasing the serum or albumin concentration in the medium increased the concentration of total 1,25-(OH)2D needed to inhibit its own production or stimulate that of 24,25-(OH)2D. In contrast, the concentration of free 1,25-(OH)2D needed to half-maximally inhibit its own production or induce 24,25-(OH)2D production remained constant at approximately 10(-11) M. We conclude that the free, not the total, 1,25-(OH)2D concentration regulates 250HD metabolism by keratinocytes, that DBPs do not facilitate 1,25-(OH)2D entry into the cell, and that these cells sense only free 1,25-(OH)2D.