The effect of temperature on proximal tubular acidification was studied in isolated rat kidney, perfused with 20 mM phosphate Ringer's containing 7.5 g/100 ml bovine albumin, equilibrated with air. Tubular pH was measured with Sb microelectrodes during stopped-flow microperfusion. The temperature of the kidney was varied between 10 and 46 degrees C. At 10 degrees C the proximal tubule was still able to maintain pH gradients of about 0.7 pH units. However, half-times (t/2) of both acidification and alkalinization were markedly increased, from 6-7 s at 37 degrees C to 27-30 s at 10 degrees C. In consequence, net H+-ion flux into the tubule was reduced to 26% of that at 37 degrees C. In this system, in the absence of exogenous HCO-3 and CO2, t/2 of acidification and alkalinization were very similar at 37 degrees C and below. Above 37 degrees C alkalinization t/2 fell markedly to 1.43 +/- 0.09 (11) s at 46 degrees C, while acidification t/2 stayed at about 7 s. H+-ion back-fluxes increased progressively from 10-46 degrees C, while secretory JH reached a maximal value at 37 degrees C and fell at higher temperatures. Apparent activation energies calculated from rate coefficients were 8.48 kcal . mol-1 for acidification, and 9.30 for alkalinization, and those calculated from JH were 6.30 and 9.55 respectively. These data indicate that both H-ion secretion and back-flux are carrier-mediated, probably flowing through the Na/H exchanger in the luminal membrane, since their activation energies are of the same order of magnitude and markedly higher than those for protons in solution.