The tubular localization of 1,25-dihydroxyvitamin D[1,25(OH)₂D₃]-stimulated calmodulin binding proteins (CaMBP-Ds) in the rat kidney and the specificity of their induction were characterized to better understand renal responses to protracted 1,25(OH)₂D₃ treatment in vivo. None of the other hormones tested (parathyroid hormone, calcitonin, estradiol-17, testosterone, progesterone, hydrocortisone, or dexamethasone) stimulated the CaMBP-Ds, whereas maximal 1,25(OH)₂D₃ stimulation occurred after a 5- to 7-day treatment with 100 ng/day 1,25(OH)₂D₃. With the exception of the more ubiquitously distributed CaMBP-D150, the CaMBP-Ds were localized in distal, but not proximal, tubule preparations. 1,25(OH)₂D₃ induction of vitamin D receptors and the CaMBP-Ds was similar with respect to dose-response and time course. Finally, the CaMBP-Ds remained elevated for at least 4 wk after 1,25(OH)₂D₃ withdrawal. Because the vitamin D-stimulated renal CaMBP-Ds are principally proteins of the distal tubule, they may be associated with renal regulation of Ca²⁺ homeostasis. The sustained induction of CaMBP-Ds is important in addressing the question of whether their induction is a function of normal Ca²⁺ homeostasis or a pathophysiological consequence of hypervitaminosis D and hypercalcemia.