1. The effect of luminal and capillary perfusion with hypotonic or hypertonic solutions containing 25 mM NaHCO3 or NaH2PO4 plus NaCl, K+, Ca2+, Mg2+ and acetate at an osmolality of 100 or 500 mosmol kg-1 on rat proximal H+ secretion was estimated by monitoring luminal pH with Sb microelectrodes. The results were compared to perfusions with the same ionic concentration in which tonicity was adjusted to 300 mosmol kg-1 with raffinose. 2. The kinetics of acidification of luminally injected bicarbonate buffer permits evaluations of H(+)-HCO3-fluxes as well as stationary pH gradients; the kinetics of alkalinization of luminally injected acid phosphate buffer indicates H(+)-HCO3-backfluxes from blood to lumen. 3. In alkalinization experiments, luminal perfusion with hypotonic solution during presence of blood in capillaries or hypotonic capillary perfusion leads to a decrease of stationary pH, an increase of alkalinization half-time and consequently a decrease of passive H(+)-HCO3-backflux. 4. In alkalinization experiments, during luminal and/or capillary perfusions with hypertonic solutions, no significant differences in the stationary pH, alkalinization half-time and H(+)-HCO3-backflux were found. 5. During acidification experiments, with both hypo- and hypertonic perfusions, no significant differences in stationary pH, acidification half-time and H(+)-HCO3-flux were observed. 6. Luminal perfusion with hypotonic solution increases specific epithelial resistance in the presence of blood in capillaries. Luminal perfusion with hypertonic solution does not change this parameter. 7. Volume changes, measured by the split-drop method, are slow during the first 30 s and do not explain the increased alkalinization half-time during luminal perfusion with hypotonic solution, since this is the period of fastest pH change. 8. Luminal perfusion with hypotonic solution decreases apparent H+ permeability in the presence of blood or hypotonic solution in capillaries. Hypertonic solutions in all experimental conditions had no significant effect on this parameter. 9. The data indicate that decrease of tonicity of fluids in contact with proximal tubule epithelium affects passive H(+)-HCO3-backflux, which proceeds in part through the shunt path, while acidification (H+ secretion), which is transcellular, is not affected by extracellular tonicity.