Studies of cortical proximal nephrons and plasma membrane vesicles suggest that a Ca-Na exchanger regulates intracellular Ca2+ concentration ([Ca2+]i) in renal tubular cells. We tested this hypothesis in isolated perfused rabbit connecting segments by measuring [Ca2+]i with fura-2. Within 2 min of replacing bath NaCl with mannitol, [Ca2+]i rose from a base line of approximately 100 nM to a peak of approximately 650 nM, then declined to a plateau of approximately 500 nM for approximately 5 min before rising to a second peak of approximately 600 nM. [Ca2+]i returned toward base line after restoring bath NaCl. Substitution of choline Cl or tetraethylammonium chloride for bath NaCl reproduced the rise in [Ca2+]i, implicating the Na+ as the mediator. Selective bath (but not lumen) Ca removal or lumen Na deletion virtually abolished these effects, suggesting that bath Na deletion causes peritubular Ca influx by a process that depends on lumen Na. Lumen Na removal lowered, whereas its repletion increased, [Ca2+]i. Smaller increments in [Ca2+]i were produced by raising lumen [Na] from 0 to 35-55 mM or from 20 to 120 mM, but not from 55 to 150 mM. Clamping bath [Ca] at approximately 100 nM abolished the rise in [Ca2+]i produced by lumen Na, corroborating the role of peritubular Ca. These results suggest a Ca influx across the basolateral membrane that is driven by a cell-to-bath [Na] gradient and that can be activated by changes in lumen [Na]. We propose that this process, in part, regulates [Ca2+]i in the rabbit connecting tubule.