The effects of fluoride on bone tissue are now well documented by in vivo histological studies performed on both human and animal bone biopsies and demonstrating an increase in osteoblast (OB) population. In order to elucidate whether the mechanism of action of fluoride on osteoblasts was direct or indirect, 14 three-week-old Sprague-Dawley rats were selected. Seven animals received 100 ppm fluoride as sodium fluoride (NaF) in drinking water for one month. The other animals, which did not receive fluoride, were considered as controls. At the end of the experiment, femurs and vertebrae were excised and osteoblastic cells were obtained after collagenase digestion separately from each animal. The osteoblastic cells derived from control and NaF-treated rats were exposed in vitro to 10 −5 M NaF. Alkaline phosphatase (AP) activity was measured, and the cellular proliferation was assessed by 3H-thymidine incorporation. Thymidine incorporation and AP activity were significantly higher in osteoblastic cells derived from NaF-treated rats than in cells obtained from control rats ( p = 0.05 and p < 0.01, respectively). In contrast, the osteoblast proliferation and activity were not modified after in vitro exposure to NaF in cells derived from control and NaF-treated rats. In conclusion, the function of osteoblasts was not modified after in vitro exposure to fluoride, In contrast, given in vivo to rats for one month, fluoride has a mitogenic effect on osteoblasts and stimulates their activity. These data emphasize the hypothesis that fluoride may act either on osteoprogenitor cells or through an indirect mechanism mediated by a cofactor.