The adaptation of the cortical collecting duct (CCD) to metabolic acidosis requires the polymerization and deposition in the extracellular matrix of the novel protein hensin. HCO₃ ^-- secreting -intercalated cells remove apical Cl ^-:HCO₃ ^- exchangers and may reverse functional polarity to secrete protons. Using intercalated cells in culture, we found that galectin-3 facilitated hensin polymerization, thereby causing their differentiation into the H ⁺-secreting cell phenotype. We examined the expression of galectin-3 in the rabbit kidney and its relation to hensin during metabolic acidosis. In control kidneys, galectin-3 was expressed in the cortical and medullary collecting ducts. In the outer cortex 26±3% of CCD cells expressed galectin-3 compared to 64±3% of the cells of the inner cortex. In the CCD, galectin-3 was rarely expressed in - intercalated cells, being primarily present in -intercalated and principal cells. During metabolic acidosis the intensity of cellular staining for galectin-3 increased and more cells began to express it; the percentage of CCD cells expressing galectin-3 increased from 26±3% to 66±3% in the outer cortex and from 64±3% to 78±4% in the inner cortex. This was particularly evident in -intercalated cells where expression was found in only 8±2% in control animals but in 75±2% during metabolic acidosis in the outer cortex and similarly for the inner cortex (26±6% to 90±7%). Importantly, during metabolic acidosis, both galectin-3 and hensin were found in the extracellular matrix of microdissected CCDs; and during acidosis many more cells exhibited this extracellular co-localization. Thus, galectin-3 may play several important roles in the CCD, including mediating the adaptation of -intercalated cells during metabolic acidosis.