The active form of vitamin D, 1,25(OH)(2)D-3, is a powerful regulator of cytosolic Ca2+-concentration ([Ca2+](i)) in a variety of cell types. The formation of 1,25(OH)(2)D-3 is inhibited by FGF23, an effect requiring presence of klotho. 1,25(OH)(2)D-3 plasma levels are excessive in klotho-deficient mice (kl/kl). A previous study revealed that klotho-deficiency is followed by decreased activation of platelets, an effect at least in part due to blunted store operated Ca2+ entry (SOCE). In other cell types 1,25(OH)(2)D-3 has been shown to up-regulate the Na+/Ca2+-exchanger, which could, depending on cell membrane potential and cytosolic Na+ concentration, either decrease or increase [Ca2+](i). The present study explored whether Na+/Ca2+-exchanger activity is different in megakaryocytes isolated from kl/kl mice than in megakaryocytes isolated from wild type mice. Na+/Ca2+-exchanger induced currents were determined by whole cell patch clamp and the Na+/Ca2+-exchanger induced alterations of [Ca2+](i) by Fura-2 fluorescence. As a result, the inward current and the increase of [Ca2+](i) following replacement of extracellular Na+ by NMDG were higher in kl/kl megakaryocytes than in wild type megakaryocytes, a difference abrogated by treatment of the mice with low Vitamin D diet. Pretreatment of wild type megakaryocytes with 1,25(OH)(2)D-3 (100 nM, 48 h) was followed by enhancement of both, inward current and increase of [Ca2+](i) following replacement of extracellular Na+ by NMDG. In conclusion, the present observations reveal a powerful stimulating effect of 1,25(OH)(2)D-3 on Na+/Ca2+-exchanger activity in megakaryocytes. (C) 2015 Elsevier Inc. All rights reserved.