The effects of bafilomycin, an inhibitor of the vacuolar-type (V-type) proton pump, on nephron ultrastructure and acidification were analyzed in isolated perfused kidneys from rats with acute metabolic acidosis. Acidic intracellular compartments were labelled with the weak base DAMP, that was subsequently visualized immunocytochemically. The distribution of the proton pump was studied by immunocytochemistry. Bafilomycin inhibited urinary acidification and caused pronounced ultrastructural changes in proximal tubule cells and B-type intercalated cells (B-IC cells). Immunoreactivity for DAMP showed that bafilomycin also inhibits intracellular acidification. The distribution of the proton pump was essentially unchanged by bafilomycin in A-IC cells. In B-IC cells, immunoreactivity accumulated over studded membrane material in the apical cytoplasm. The results indicate that the V-type proton pump is important both for intracellular and for urinary acidification.