Vitamin D metabolites exert both acute and chronic influences on proximal tubule function. To further evaluate vitamin D action on the kidney, we examined the immediate effects of vitamin D metabolites on cytoplasmic calcium ion concentration [( Ca2+]i), using fura-2 and patch-clamp method in cultured proximal straight tubule cells of rabbit kidney. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] and 25-hydroxyvitamin D3 [25(OH)D3] evoked a transient rise in [Ca2+]i, and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] caused a sustained rise in [Ca2+]i; all effects were dose dependent. [Ca2+]i transient, evoked by 1,25(OH)2D3 alone, was abolished in Ca(2+)-free media. Pretreatment of cells in Ca(2+)-free media with caffeine (4 mM) or ryanodine (1 microM) to deplete Ca2+ store of endoplasmic reticulum or with TMB-8 (5 mM) to block Ca2+ release from storage blunted the effect of 25(OH)D3 on [Ca2+]i but not of 24,25(OH)2D3. Data were also supported by activities of Ca-dependent K channel and show that these three vitamin D metabolites in pharmacological doses increase [Ca2+]i of proximal tubule cells from different sources.