Changes in intracellular pH (pHi) were measured using the pH indicator, BCECF, in principal cells from split opened cortical collecting tubules (CCTs) derived from rabbits maintained on a normal diet. This monolayer preparation has the advantage of allowing us to visualize the morphological differences in the two major cell types in this nephron segment under transmitted light. The visual identification of the cell types was verified using emission measurements taken from single principal and intercalated cells in the opened tubule which had been exposed to fluorescein isothiocyanate (FITC)-labeled peanut lectin. We confirmed the existence of an amiloride-sensitive Na/H exchange process activated during intracellular acidosis in principal cells. In addition, the exchanger was active under basal conditions and over a wide range of pHi. Because the exchanger was active under basal conditions we tested the hypothesis that changes in intracellular Na (Nai) would alter pHi in a predictable way. Maneuvers designed to alter Nai were without significant effects within a 10-min time frame. Specifically, addition of 100 microM ouabain to increase Nai or exposure of the tubules to 10(-5) M amiloride to decrease luminal Na entry and reduce Nai did not have an effect on pHi. In some experiments we did observe however, after a 30-min exposure to ouabain, a small decrease in pHi. These results suggest that Na/H exchange is a major regulator of pHi in principal cells. However, regulation of Na transport by changes in pHi in principal cells of rabbit CCT via the activity of a Na/H exchanger do not seem to contribute to the feedback control of Na transport.