We recently showed that indapamide (IDP), a thiazide-related diuretic, increases bone mass and decreases bone resorption in spontaneously hypertensive rats supplemented with sodium. In the present study, we evaluated the in vitro effects of this diuretic on bone cells, as well as those of hydrochlorothiazide (HCTZ), the reference thiazide, and acetazolamide (AZ), a carbonic anhydrase (CA) inhibitor. We showed that 10(-4) M IDP and 10(-4) M AZ, as well as 10(-5) M pamidronate (APD), decreased bone resorption in organ cultures and in cocultures of osteoblast-like cells and bone marrow cells in the presence of 10(-8) M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We investigated the mechanism of this antiresorptive effect of IDP; IDP decreased osteoclast differentiation as the number of osteoclasts developing in coculture of marrow and osteoblast-like cells was decreased markedly. We then investigated whether IDP affected osteoblast-like cells because these cells are involved in the osteoclast differentiation. Indeed, IDP increased osteoblast-like cell proliferation and alkaline phosphatase (ALP) expression. Nevertheless, it did not modify the colony-stimulating factor 1 (CSF-1) production by these cells. In addition, osteoblast-like cells expressed the Na+/Cl- cotransporter that is necessary for the renal action of thiazide diuretics, but IDP inhibited bone resorption in mice lacking this cotransporter, so the inhibition of bone resorption and osteoclast differentiation did not involve this pathway. Thus, we hypothesized that IDP may act directly on cells of the osteoclast lineage. We observed that resorption pits produced by spleen cells cultured in the presence of soluble osteoclast differentiation factor (sODF) and CSF-1 were decreased by 10(-4) M IDP as well as 10(-5) M APD. In conclusion, in vitro IDP increased osteoblast proliferation and decreased bone resorption at least in part by decreasing osteoclast differentiation via a direct effect on hematopoietic precursors.