Cortical thick ascending limbs of Henle's loop were dissected from the kidneys of chronically vitamin D-deficient or -replete rabbits and perfused in vitro. Unidirectional transepithelial calcium fluxes from lumen to bath were measured with 45Ca. The tubules were bathed in a solution containing 150 mM sodium and perfused with a solution containing 60 mM sodium to simulate conditions in the cortical thick ascending limb in vivo. Transepithelial voltages were equal across tubules from vitamin D-deficient and -replete rabbits. Likewise, baseline and parathyroid hormone-stimulated calcium fluxes were the same in tubules from the two groups. Because calcidiol and calcitriol were undetectable in the serum of the vitamin D-deficient rabbits, we suggest that neither of these endogenous vitamin D metabolites is essential in the regulation of calcium absorption in this portion of the rabbit nephron.