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-(0H)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) 1α-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. (Endocrinology124: 649–654, 1989)