Apical low-conductance SK and high-conductance Ca²⁺-activated BK channels are present in distal nephron, including the cortical collecting duct (CCD). Flow-stimulated net K secretion (JK) in the CCD is (i) blocked by iberiotoxin, an inhibitor of BK but not SK channels, and (ii) associated with an increase in [Ca²⁺]_i, leading us to conclude that BK channels mediate flow-stimulated JK. To examine the Ca²⁺-dependence and sources of Ca²⁺ contributing to flow-stimulated JK, JK and net Na absorption (JNa) were measured at slow (~1) and fast (~5 nl/min.mm) flow rates in rabbit CCDs microperfused in the absence of luminal Ca²⁺ or after pretreatment with BAPTA-AM to chelate intracellular Ca²⁺, 2-aminoethoxydiphenyl borate (2-APB) to inhibit the inositol 1,4,5-trisphosphate (IP₃) receptor, or thapsigargin to deplete internal stores. These treatments, which do not affect flow-stimulated JNa (Morimoto et al, AJP 291:F663-9, 2006), inhibited flow-stimulated JK. Increases in [Ca²⁺]_i stimulate exocytosis. To test whether flow induces exocytic insertion of preformed BK channels into the apical membrane, CCDs were pretreated with 10 µM colchicine (COL) to disrupt microtubule function or 5 µg/ml brefeldin-A (BFA) to inhibit delivery of channels from the intracellular pool to the plasma membrane. Both agents inhibited flow-stimulated JK but not JNa (Morimoto et al, AJP 291:F663-9, 2006), although COL but not BFA also blocked the flow-induced [Ca²⁺]_i transient. We thus speculate that BK channel-mediated flow-stimulated JK requires an increase in [Ca²⁺]_i due in part to luminal Ca²⁺ entry and ER Ca²⁺ release, microtubule integrity, and exocytic insertion of preformed channels into the apical membrane.