In vivo effects of basic fibroblast growth factor (bFGF) on bone formation was examined in rats. Daily systemic injections of 100 micrograms/kg bFGF for 7 days caused a marked stimulation of endosteal bone formation in both cortical and secondary cancellous bone areas. Histological examinations revealed that the sequence of responses to the injections of bFGF consisted of three phases: an early increase in the number of preosteoblastic cells over the osteoblastic cell layer (days 1-3), recruitment of osteoblasts from preosteoblastic cells (days 3-5), and an increase in new bone formation (days 5-7). These histological changes in the endosteum correlated closely with histomorphometrical parameters of bone formation, and the endosteal mineral apposition rate was almost unaffected during the initial 4 days but was markedly enhanced after this period. Immunohistochemical examinations using antitransforming growth factor (TGF)-beta 1 antibody demonstrated that immunostaining of preosteoblastic cells for TGF-beta already increased 1 day after bFGF treatment. Distribution of TGF-beta in osteoblasts and bone matrices began to increase on day 3, and all the osteoblasts and new bone matrices were intensively immuno-stained on day 7. These results demonstrate that systemic injections of bFGF in rats stimulate endosteal bone formation, and that the stimulation of bone formation is preceded by an initial increase in preosteoblastic cells with later recruitment of osteoblasts from these cells. Because the distribution of TGF-beta in the endosteal cells is increased by bFGF, the effect of bFGF may at least in part be mediated by TGF-beta. However, the precise mechanism of action of bFGF on bone formation remains to be clarified.