Thiazide diuretics are commonly used for the treatment of hypertension and their use has been associated with an increased bone mineral density in patients undergoing such therapy. Their molecular target is the thiazide-sensitive sodium-chloride cotransporter (NCC), which is expressed in the kidney distal tubule. It has been proposed that, by inhibiting the kidney NCC, thiazides produce a positive calcium balance and drive the observed bone-sparing effect. However, a direct skeletal effect of thiazides and the expression of NCC in bone cannot be ruled out and, indeed, has been hypothesized. We have previously reported the presence of NCC mRNA and protein in cells of the osteoblast lineage in freshly frozen undecalcified preparations of human and rat bone, as well as in tissue culture models of osteoblasts, the bone-forming cells [1]. Here we investigated the effect of metolazone, a thiazide-like diuretic, on the differentiation of human osteoblast-derived cells (MG63) and on fetal rat calvarial cells (FRC), a model for osteoblasts, and detected the consequence of an NCC overexpression on FRC differentiation. Our results indicate that chronic metolazone treatment (1-100 μM) dose-dependently promoted the expression of osteoblast differentiation markers, Runx2 and osteocalcin, measured by western blotting, in the absence of a proliferative effect (n=3; p<0.05 by ANOVA, Tukey post hoc test). This effect was specific, as the expression of an early differentiation marker, type I collagen, was not affected. In FRC cells, metolazone dose-dependently increases the formation of mineralized nodules, quantified by von Kossa staining (n=6; p<0.05 by ANOVA, Tukey post hoc test). These findings are specific to thiazide diuretics and are not mimicked by the loop diuretic bumetanide. Finally, overexpression of NCC in FRC cells significantly increased metolazone-induced mineralization (n=3; p<0.05 by ANOVA, Tukey post hoc test). Taken together, these results provide strong evidence for a direct action of thiazides on bone, through NCC, an effect which is enhanced by NCC overexpression. Our findings support the view that thiazides should be considered as diuretics of choice in elderly, osteoporotic patients.