To examine the mechanism of preferential luminal ammonia secretion in the proximal tubule the apical and basolateral membrane pathways for NH3 and NH4+ movement were studied in the in vivo microperfused rat proximal tubule. Na and Cl were absent from all perfusates. Changes in pHi in response to rapid addition of NH3-NH4+ to either the luminal or peritubular perfusates were measured microfluorimetrically and expressed as the H(+)-equivalent flux (JeqH in pmol.mm-1.min-1). After ammonia addition ([NH3] 0.3 mM; [NH4+] 14.7 mM) to the luminal or peritubular fluids, pHi increased, and JeqH = 1,713 +/- 181 and 1,040 +/- 132 pmol.mm-1.min-1, respectively. To determine whether the above difference was due to NH3- or NH4(+)-driven fluxes, the effect of a fivefold greater [NH4+] ([NH3] 0.3 mM; [NH4+] 74.5 mM) on JeqH was examined. With luminal addition of a fivefold greater [NH4+], JeqH increased to 3,299 +/- 292 pmol.mm-1.min-1, demonstrating a pathway for NH4(+)-driven H+ efflux. One millimolar luminal amiloride inhibited JeqH in response to luminal NH3-NH4+ addition, suggesting that the amiloride-sensitive Na(+)-H+ antiporter mediates the NH4(+)-driven H+ efflux. JeqH was unaffected by addition of a fivefold greater [NH4+] to the peritubular perfusate, demonstrating the absence of an NH4(+)-driven H+ flux on the basolateral membrane. From these data, the calculated NH3 permeabilities were 6.2 +/- 1.3 and 7.0 +/- 0.9 X 10(-2) cm/s for the apical and basolateral membranes, respectively (NS). We conclude that apical and basolateral membrane NH3 permeabilities are similar and large. The apical membrane can also transport NH4+ on the amiloride-sensitive Na(+)-H+ antiporter.