To explore biochemical and functional differences between principal (PC) and intercalated cells (ICC), we have developed a method for separating them from rabbit kidney. Fragments of cortical collecting ducts were isolated by immunodissection, and single cells obtained from these clusters were stained with fluorochrome-conjugated, cell-specific markers. PC and ICC were then separated by fluorescence-activated cell sorting. Identity of the sorted cells was confirmed by staining with other cell-specific monoclonal antibodies (MCABs) or peanut lectin. Purity was greater than 99% for ICC and greater than 96% for PC. Arginine vasopressin (AVP) increased adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in both cell types, but maximal stimulation was significantly greater with PC (approximately 20-fold) than with ICC (2.7-fold). Half-maximal stimulation was seen at approximately 2 x 10(-10) M AVP with both cell types. Isoproterenol increased cAMP levels only with ICC (from 1.23 +/- 0.16 to 12.06 +/- 1.25 fmol/cell; P less than 0.001). The number of ouabain binding sites and the activity of Na+-K+-ATPase was significantly higher in sorted PC than ICC (2.2 X 10(6) vs. 9.6 X 10(5) binding sites; 19.2 vs. 9.6 fmol.min-1.cell-1 ATP hydrolyzed in PC vs. ICC, respectively). These results demonstrate the feasibility of isolating homogeneous populations of PC and ICC, which is useful for further studies of their biochemical and functional characterization.