In previous studies we have shown that clinically effective concentrations of fluoride (5 to 30 μmol/L) could also have direct effects in vitro on skeletal tissues to increase embryonic chick bone formation and bone cell proliferation ( 3[H]-thymidine incorporation into DNA). From these observations, we hypothesized that fluoride-stimulated bone formation might be mediated by a direct effect of fluoride to increase bone cell proliferation. The current studies were intended to investigate the mechanism of fluoride-stimulated 3[H]-thymidine incorporation, in chick calvarial cell cultures, by assessing mitogenic interactions between fluoride and inorganic phosphate, bone-derived growth factors, and systemic skeletal effectors. With respect to fluoride-phosphate interactions, the results of our studies indicate that the effect of fluoride was dependent on the phosphate concentration in the medium. Fluoride did not increase 3[H]-thymidine incorporation in BGJ b medium containing 1 mmol/L (total) phosphate; but, in 1.6 mmol/L phosphate medium, fluoride caused a dose-dependent increase in 3[H]-thymidine incorporation, between 1 and 20 μmol/L ( P<.001). The action of fluoride was also dependent on the presence of a bone cell mitogen. Fluoride increased 3[H]-thymidine incorporation when added to calvarial cell cultures in the cell-conditioned medium, but had no effect in unconditioned (ie, fresh) medium. The action of fluoride could be restored by adding an exogenous growth factor (ie, concentrated cell-conditioned medium, bone-derived growth factors, or a systemic bone cell mitogen) to the unconditioned culture medium, P<.05 for each effector. All of the systemic bone cell mitogens we tested (insulin, IGF-1, PTH, and calcitonin) increased the maximum response to fluoride ( P <.05), and, conversely, fluoride increased the maximum response to each of these effectors. The activations were noncompetitive. (1,25-dihydroxy-vitamin D decreased 3[H]-thymidine incorporation, with or without added fluoride.) Finally, our data showed that the activity of fluoride was dependent on the presence of mitogen-responsive osteoprogenitor cells. Using calvarial cell cultures enriched for differentiated osteoblasts and for osteoprogenitor cells, we found an inverse correlation between flouride-stimulated 3[H]-thymidine incorporation and alkaline phosphatase activity per milligram of cell protein ( r = −0.88, P < .05). Together, these data demonstrate that fluoride-stimulated 3[H]-thymidine incorporation can be modulated by systemic skeletal effectors, is dependent on the phosphate concentration in the medium, and requires both a bone cell mitogen and mitogen-responsive osteoprogenitor cells. With regard to mechanism, our data indicate that fluoride increases bone cell proliferation by enhancing the activity of bone cell mitogens.