Studies in our laboratory have demonstrated total CO2 absorption (JtCO2) and total ammonia secretion in the terminal inner medullary collecting duct (tIMCD) perfused in vitro. The purpose of the present study was to determine whether the H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) participates in proton secretion or JtCO2 in this segment. Tubules from the middle third of the tIMCD were dissected from rats with chronic metabolic acidosis (300 mM NH4Cl, 3-4 days in drinking water) and perfused in vitro. Perfusate and bath were symmetrical solutions containing 5 mM KCl, 6 mM NH4Cl, and 25 mM NaHCO3. Bafilomycin A1 (5 nM), a specific inhibitor of the H(+)-ATPase, did not affect JtCO2 compared with baseline (JtCO2, 3.0 +/- 1.0 and 3.0 +/- 0.8; n = 6, P = not significant) or with time controls (n = 4). With removal of luminal K+, JtCO2 fell from 2.8 +/- 0.6 to 1.6 +/- 0.4 pmol.mm-1.min-1 (n = 5, P < 0.05). To further evaluate K(+)-sensitive JtCO2, the effect of H(+)-K(+)-ATPase inhibition on JtCO2 was explored using the specific H(+)-K(+)-ATPase inhibitor, Sch-28080. Addition of 10 microM Sch-28080 to the luminal perfusate decreased JtCO2 (2.7 +/- 0.4 to 1.4 +/- 0.5 pmol.mm-1. min-1; n = 5, P < 0.05) but did not alter transepithelial membrane potential. Thus luminal Sch-28080 addition, as well as luminal K+ removal, limits apical H+ exit or OH-/HCO3- entry. These results demonstrate that net acid secretion is mediated by the H(+)-K(+)-ATPase in the tIMCD.