Cells from the inner medullary collecting duct (IMCD) exhibit Na(+)-H+ exchange. The present studies were performed to address certain important characteristics of this process in cultured IMCD cells. First, Na(+)-H+ exchange was found to be present both at 37 degrees C and at 25 degrees C, in contrast to Na(+)-independent H+ extrusion, which was only observed in some cultures and only at 37 degrees C. Second, with the use of image analysis techniques, virtually all cells in IMCD cultures were demonstrated to possess Na(+)-H+ exchange, whether or not the cells exhibited Na(+)-independent intracellular pH recovery from acid loads. Also, Na(+)-H+ exchange was found to be expressed on the basolateral aspect of these cells, but not on the apical membrane. These properties of IMCD Na(+)-H+ exchange are consistent with a function to regulate intracellular pH rather than mediate transepithelial acid-base transport. Na(+)-H+ exchange in IMCD cells was also compared with that in cultured renal proximal tubule cells. Despite physiologically distinct roles in vivo, Na(+)-H+ exchange in these two cell types in culture was found to be similar with respect to the Km for Na+ and the Ki for 5-(N-ethyl-N-isopropyl)amiloride. These data are consistent with functionally similar (if not identical) processes mediating Na(+)-H+ exchange in these two cell types, but with opposite polarity.