These studies were performed to determine whether in vitro elevation of bath PCO2 with associated reduction in pH (acute respiratory acidosis) affected bicarbonate transport by isolated perfused rabbit cortical collecting tubules (CCT) and collecting tubules from the outer (OMCTos) and inner (OMCTis) stripes of the outer medulla. When the PCO2 was elevated and pH reduced from approximately 7.4 to 7.0 the rate of total CO2 absorption increased to 252% of that observed at pH 7.4 in CCT, 146% in OMCTos, and 150% in OMCTis. In OMCTis, pretreatment with colchicine inhibited the stimulation of total CO2 absorption associated with respiratory acidosis, whereas lumicolchicine did not. Similar inhibition was observed in the presence of maptam and a low calcium concentration and in the presence of a calmodulin inhibitor. No differences were observed in apical or basolateral membrane morphometry of principal or intercalated cells between control tubules and those subjected to respiratory acidosis. The results indicate that acute respiratory acidosis stimulates acidification by the rabbit distal nephron in vitro through a process(es) that, at least in OMCTis, evidently involves the cell cytoskeleton and changes in cell calcium and calmodulin activities.