The early distal tubule (EDT) of the frog nephron, like the thick ascending limb in mammals, mediates the transepithelial absorption of NaCl. The continued absorption of NaCl in the face of varying Na⁺ load is maintained by coordination of the activity of ion transporting proteins in the apical and basolateral membranes - so-called pump-leak coupling. Previous studies have identified intracellular Ca²⁺, originating from an intracellular Ca²⁺ store, as playing a key role in pump leak coupling in the EDT. The purpose of the experiments described in this paper was to identify the intracellular Ca²⁺ storage pools in the renal diluting segment. Store Ca²⁺ movements were monitored by the fluorescence of mag-fura-2 in permeabilised segments of frog EDTs. The presence of both ATP and Ca²⁺ were required to maintain store Ca²⁺ content. Removal of either of these substrates resulted in a passive leak of Ca²⁺ from the stores. The uptake of Ca²⁺ into the store was sensitive to the SERCA inhibitor TBQ, whilst Ca²⁺ release from the store was stimulated by IP₃, but not cADPR. Store Ca²⁺ was insensitive to the mitochondrial ATP-synthase inhibitor oligomycin, and, under conditions that energised _m, the complex 1 inhibitor rotenone and the protonophore FCCP. Ionomycin was able to mobilise store Ca²⁺ following exposure to IP₃. These results suggest that the ER is a dominant Ca²⁺ store in the frog EDT. A second pool, sensitive to ionomycin but not IP₃, may overlap with the IP₃-sensitve pool. The data also rule out any contribution by mitochondria to EDT Ca²⁺ cycling.