In vivo microperfusion studies show augmented proton secretion in the distal tubule of rats with chronic metabolic alkalosis. The present studies used the same technique to determine whether this augmented proton secretion is due predominantly to an increase in substrate affinity or alternatively to a predominant increase in the maximal proton secretory rate. Surface distal tubules of alkalotic and control rats were microperfused in vivo with solutions containing increasing concentrations of HCO3. Proton secretion was determined as the difference between measured net HCO3 reabsorption and passive HCO3 transport calculated by use of the permeability derived from perfusing with a HCO3-free solution. Proton secretion was expressed as a function of the initial luminal HCO3 concentration and was assumed to follow saturable Michaelis-Menten kinetics. Alkalotic animals had a significantly higher Km (33.9 vs. 21.6 mM, P less than 0.03) and Vmax (223.8 vs. 99.1 pmol.mm-1.min-1, P less than 0.001) compared with control. These data are consistent with the augmented proton secretion in the distal tubule of alkalotic animals as predominantly due to an increased maximal proton secretory rate rather than to increased substrate affinity.