Metabolic acidosis in vivo, as well as in vitro (1 h at pH 6.8 followed by 2 h at pH 7.4) stimulates H⁺-ATPase-dependent H⁺ secretion in outer medullary collecting ducts from the inner stripe (OMCD_i) (S. Tsuruoka and G. J. Schwartz. J. Clin. Invest. 99: 1420–1431, 1997). Another group has shown that the adaptation to metabolic acidosis in vivo is mediated by an apical polarization of H⁺ pumps without an increase in total H⁺pump mRNA or protein (B. Bastani, H. Purcell, P. Hemken, D. Trigg, and S. Gluck. J. Clin. Invest. 88: 126–136, 1991). To further address the mechanism of adaptation, we measured net absorption before and after applying protein/RNA synthesis and signal transduction inhibitors during the 1 h of low pH and a cytoskeletal inhibitor during the entire 3-h incubation. Net transport, measured by microcalorimetry, increased ∼33% after in vitro acidosis. This increase was prevented by application during the first hour of anisomycin (10 μM) or actinomycin D (4 μM), but not by anisomycin applied during the 2-h incubation at pH 7.4. Similar results were obtained with the cell calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM, 20 μM), the calmodulin antagonist, calmidazolium (30 nM), the endoplasmic reticulum Ca-ATPase inhibitor, thapsigargin (100 nM), and the protein kinase C (PKC) inhibitor, staurosporine (100 nM), applied during the 1 h at pH 6.8, but not with BAPTA-AM or thapsigargin used during the 2-h incubation at pH 7.4. Colchicine (10 μM) applied during the entire 3-h incubation also prevented this adaptive increase in H⁺ secretion, whereas lumicolchicine (10 μM, the inactive congener) did not. Colchicine also reversibly prevented any adaptive increases in transepithelial positive voltage. Thus the adaptation to acidosis in vitro required RNA and protein synthesis, changes in intracellular calcium and PKC activity, and intact microtubules. Time was required for the adaptation to occur, as the increase in transport was small after <3-h incubation. Protein synthesis and changes in cell calcium were critical during the initial period of low pH but not once the acid stimulus had been removed. Exocytosis of H⁺ pumps appears to occur continually during the entire 3-h incubation. These data would suggest that the synthesis and regulation of proteins involved in shuttling H⁺ pumps in cytoplasmic vesicles to the apical membrane via exocytosis are important for the OMCD_i to adapt to low pH in vitro and probably to metabolic acidosis in vivo.