Nucleotide binding to purinergic P2 receptors contributes to the regulation of a variety of physiological functions in renal epithelial cells. Whereas P2 receptors have been functionally identified at the basolateral membrane of the cortical collecting duct (CCD), a final regulatory site of urinary Na⁺, K⁺, and acid-base excretion, controversy exists as to whether apical purinoceptors exist in this segment. Nor has the distribution of receptor subtypes present on the unique cell populations that constitute Ca²⁺ the CCD been established. To examine this, we measured nucleotide-induced changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in fura 2-loaded rabbit CCDs microperfused in vitro. Resting [Ca²⁺]_i did not differ between principal and intercalated cells, averaging ∼120 nM. An acute increase in tubular fluid flow rate, associated with a 20% increase in tubular diameter, led to increases in [Ca²⁺]_i in both cell types. Luminal perfusion of 100 μM UTP or ATP-γ-S, in the absence of change in flow rate, caused a rapid and transient approximately fourfold increase in [Ca²⁺]_i in both cell types (P< 0.05). Luminal suramin, a nonspecific P2 receptor antagonist, blocked the nucleotide- but not flow-induced [Ca²⁺]_i transients. Luminal perfusion with a P2X (α,β-methylene-ATP), P2X₇ (benzoyl-benzoyl-ATP), P2Y₁ (2-methylthio-ATP), or P2Y₄/P2Y₆ (UDP) receptor agonist had no effect on [Ca²⁺]_i. The nucleotide-induced [Ca²⁺]_i transients were inhibited by the inositol-1,4,5-triphosphate receptor blocker 2-aminoethoxydiphenyl borate, thapsigargin, which depletes internal Ca²⁺ stores, luminal perfusion with a Ca²⁺-free perfusate, or the L-type Ca²⁺ channel blocker nifedipine. These results suggest that luminal nucleotides activate apical P2Y₂ receptors in the CCD via pathways that require both internal Ca²⁺mobilization and extracellular Ca²⁺ entry. The flow-induced rise in [Ca²⁺]_i is apparently not mediated by apical P2 purinergic receptor signaling.