Studies on adenylate cyclase response of the nephron fragments to hormones and drugs have suggested that there is a functionally distinct segment tentatively called the connecting tubule (CNT), which is located between the distal (DT) and the cortical collecting tubule (CCT). The functional significance of these biochemical findings was examined with isolated rabbit renal tubules perfused in vitro. The transepithelial voltage (PDt) of the DT, CNT, and CCT were, respectively, -28.7 +/- 3.24 mV (24), -27.0 +/- 2.69 mV (24), and -3.5 +/- 2.14 mV (11) in the normal rabbits. The PDt of the CCT increased to -32.2 +/- 2.02 mV (33) when rabbits were pretreated for at least 3 days with deoxycorticosterone acetate, DOCA (1 mg/kg/day, i.m.), whereas the PDt of the DT and the CNT remained unchanged. The PDt of the CCT obtained from deoxycorticosterone acetate- (DOCA) treated animals decreased after addition of antidiuretic hormone (ADH) or isoproterenol (ISO) to the bath. The PDt of the CNT also responded to these agents, but the dose required to obtain the same response was quite different: The CNT was 100-fold more sensitive to ISO as compared to the CCT, whereas the CCT was 10-fold more sensitive to ADH than was the CNT. In contrast, the PDt of the DT did not respond to any of these agents, even at a higher concentration. After addition of ADH (200 microU/ml) to the bath, the osmotic water permeability (10(-8) cm2 . sec-1 . atm-1) of the CCT increased from 1.13 +/- 0.83 to 7.46 +/- 2.36, but that of the CNT remained low (0.36 +/- 0.78 in control vs. 0.48 + 0.64 after ADH). These observations support the view that the CNT is functionally distinct from either the DT or the CCT.