Carbonic anhydrase II (CA II) expression is characteristic for the early stage of osteoclast differentiation. To study how CA II, which is crucial in proton generation in mature osteoclasts, influences the osteoclast differentiation process we performed rat bone marrow cultures. In this model, acetazolamide, a specific CA inhibitor, decreased the 1,25(OH)₂D₃-induced formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells, in a dose-dependent manner. We then performed intracellular pH (pH_i) and Ca²⁺(Ca²⁺_i) measurements for cultured osteoclasts and noticed that addition of acetazolamide caused a rapid, transient increase of both parameters. The increase in pH_iwas dependent neither on the culture substrate nor on the extracellular pH (pH_e) but the increase could be diminished by DIDS or by bicarbonate removal. Membrane-impermeable CA inhibitors (benzolamide and pd5000) did not have this effect. Addition of CA II antisense oligonucleotides into the cultures reduced the pH_iincrease significantly. CA II inhibition was also found to neutralize the intracellular vesicles at extracellular pH (pH_e) of 7.4, but at less extent at pH_e7.0. In mouse calvaria cultures, bone resorption was inhibited dose dependently by acetazolamide at pH_e7.4 while inhibition was smaller at pH_e7.0. We conclude that CA II is essential not only in bone resorption but also in osteoclast differentiation. In both processes, however, the crucial role of CA II is at least partially due to the effect on the osteoclast pH_iregulation.