In kidney medulla, intercalated cells (ICs) all have an apical proton-pumping ATPase, but in the cortex, ICs with apical (type A cells) and basolateral (type B cells) proton pumps coexist. Proton pumps in proximal-tubule epithelial cells are located in a band of apical membrane at the base of the brush border. To determine the effect of microtubule disruption on proton-pump polarity in these cell types, we immunolocalized proton pumps in normal and colchicine-treated rat kidneys. In addition, NEM-sensitive ATPase was measured in brush-border (BBMV) and basolateral membrane vesicles from kidney cortex. Rats were injected with colchicine, and kidneys were fixed 4-24 h later. In both cortical and medullary IC, proton pumps were localized on vesicles that were scattered throughout the cell. A- and B-type ICs were no longer distinguishable on the basis of proton-pump polarity. In the medulla, no concentration of proton pumps at the basolateral pole of ICs was detectable. In proximal tubules, pumps were also scattered on vesicles throughout the cytoplasm. NEM-sensitive ATPase was reduced by over 60% in BBMV, and was also reduced in basolateral membranes, indicating no increased insertion of NEM-sensitive ATPase into basolateral membranes after colchicine treatment. We conclude that a change in the polymerization state of microtubules is not sufficient to account for the differential targetting of proton pumps in subpopulations of intercalated cells.