Extracellular acidification by osteoclasts is essential to bone resorption. During proton pumping, intracellular pH (pH_i) is thought to be kept at a near-neutral level by chloride/bicarbonate exchange. Here we show that the Na⁺-independent chloride/bicarbonate anion exchanger 2 (Ae2) is relevant for this process in the osteoclasts from the long bones of Ae2_a,b^-/- mice (deficient in the main isoforms Ae2a, Ae2b₁, and Ae2b₂). Although the long bones of these mice had normal numbers of multinucleated osteoclasts, these cells lacked a ruffled border and displayed impaired bone resorption activity, resulting in an osteopetrotic phenotype of long bones. Moreover, in vitro osteoclastogenesis assays using long-bone marrow cells from Ae2_a,b^-/- mice suggested a role for Ae2 in osteoclast formation, as fusion of preosteoclasts for the generation of active multinucleated osteoclasts was found to be slightly delayed. In contrast to the abnormalities observed in the long bones, the skull of Ae2_a,b^-/- mice showed no alterations, indicating that calvaria osteoclasts may display normal resorptive activity. Microfluorimetric analysis of osteoclasts from normal mice showed that, in addition to Ae2 activity, calvaria osteoclasts—but not long-bone osteoclasts—possess a sodium-dependent bicarbonate transporting activity. Possibly, this might compensate for the absence of Ae2 in calvaria osteoclasts of Ae2_a,b^-/- mice.—Jansen, I. D. C., Mardones, P., Lecanda, F., de Vries, T. J., Recalde, S., Hoeben, K. A., Schoenmaker, T., Ravesloot, J.-H., van Borren, M. M. G. J., van Eijden, T. M., Bronckers, A. L. J. J., Kellokumpu, S., Medina, J. F., Everts, V., Oude Elferink, R. P. J. Ae2_a,b-deficient mice exhibit osteopetrosis of long bones but not of calvaria.