We examined changes in electrical and morphological properties of rat osteoclasts in response to prostaglandin E₂ (PGE₂). PGE₂ (>10 nM) stimulated an outwardly rectifying Cl^- current in a concentration-dependent manner and caused a long-lasting depolarization of cell membrane. This PGE₂-induced Cl^- current was reversibly inhibited by 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB) and tamoxifen. The anion permeability sequence of this current was I^- > Br^- Cl^- > gluconate^-. When outwardly rectifying Cl^- current was induced by hyposmotic extracellular solution, no further stimulatory effect of PGE₂ was seen. Forskolin and dibutyryl adenosine 3',5'-cyclic monophosphate (db-cAMP) mimicked the effect of PGE₂. The PGE₂-induced Cl^- current was inhibited by pretreatment with guanosine 5'-O-2-thiodiphosphate (GDPS), Rp-adenosine-3',5'-cyclicmonophosphothioate (Rp-cAMPS), N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide dihydrochloride (H-89) and protein kinase A inhibitors. Even in absence of non-osteoclastic cells, PGE₂ (1 µM) reduced cell surface area and suppressed motility of osteoclasts, and these effects were abolished by Rp-cAMPS or H-89. PGE₂ is known to exert its effects through four subtypes of PGE receptors (EP1~EP4). The EP2 and EP4 agonists (ONO-AE1-259 and ONO-AE1-329, respectively), but not EP1 and EP3 agonists (ONO-DI-004 and ONO-AE-248, respectively) mimicked the electrical and morphological actions of PGE₂ on osteoclasts. Our results show that PGE₂ stimulates rat osteoclast Cl^- current by activation of a cAMP-dependent pathway through EP2 and, to a lesser degree, EP4 receptors and reduces osteoclast motility. This effect is likely to reduce bone resorption.