By cable analysis and intracellular microelectrode impalement in the in vitro perfused renal tubule, we identified α- and β-intercalated (IC) cells along the rabbit distal nephron segments, including the connecting tubule (CNT), the cortical collecting duct (CCD), and the outer medullary collecting duct in the inner stripe (OMCD(i)). IC cells were distinguished from collecting duct (CD) cells by a relatively low basolateral membrane potential (V(B)), a higher fractional apical membrane resistance, and apparent high Cl- conductances of the basolateral membrane. Two functionally different subtypes of IC cells in the CCD were identified based on different responses of V(B) upon reduction of the perfusate Cl- from 120 to 12 mM: the basolateral membrane of β-IC cells was hyperpolarized, whereas that of α-IC cells was unchanged. This is in accord with the hypothesis that the apical membrane of β-IC cells contains some Cl--dependent entry processes, possibly a Cl-/HCO3- exchanges. Further characterization of electrical properties of both subtypes of IC cells were performed upon lowering bath or perfusate Cl- from 120 to 12 mM , and raising bath or perfusate K+ from 5 to 50 mM. A 10-fold increase in the perfusate K+ had no effect on V(B) in both subtypes of IC cells. Upon abrupt changes in Cl- or K+ concentration in the bath, a large or a small depolarization of the basolateral membrane, respectively, was observed in both subtypes of IC cells. The electrical properties of α- and β-IC cells were similar among the distal nephron segments, but their distribution was different: in the CNT which consists of IC cells and CNT cells, 97.3% (36/37) of IC cells were of the β type. In the CCD, which consists of IC cells and CD cells, 79.8% (79/99) of IC cells were of the β-type, whereas in the OMCD(i) 100% (19/19) were of the α type, suggesting that the β type predominates in the earlier and the α type in the later segment.