In this study we defined some of the important elements in the acidification process of rat inner medullary collecting duct (IMCD) cells in culture. After cell acidification, i.e., cell pH (pHi) = 6.51 +/- 0.02, pHi increased 0.046 +/- 0.003 units/min. N-ethylmaleimide, N,N'-cyclohexylcarbodiimide, and bafilomycin reduced this rate by over 85%. In contrast, omeprazole and Sch-28080 had no effect. 1,2-Bis(2-aminophenoxy)ethane-N,N,N,N'-tetraacetic acid, which prevents a rise in cell Ca2+ concentration ([Ca2+]i) reduced the rate of pHi recovery to 0.013 +/- 0.002 units/min. Calmodulin inhibitors or disruption of cytoskeletal elements with cytochalasin B and colchicine also reduced pHi recovery significantly. In addition, these cells contain acidic vesicles and undergo pHi-regulated endocytosis and exocytosis, which are inhibited by disrupting the cytoskeleton. We conclude that, in our cultured line of rat IMCD cells, proton secretion is mediated by an H(+)-adenosinetriphosphatase. Changes in pHi produce alterations in acid secretion through a signal cascade that requires changes in [Ca2+]i, activation of calmodulin, an intact cytoskeleton, and alteration in the rate of exocytosis and endocytosis.