The insulin-like growth factor (IGF) system plays an important role in the autocrine and paracrine regulation of bone formation and remodeling. The aim of this study was to evaluate the role of the autocrine IGF system during osteogenic differentiation in rat tibial osteoblasts (ROB) in culture. In this in vitro model, the stages of osteogenesis studied were S1, corresponding to the onset of alkaline phosphatase (AP) expression (days 0-3); S2, coincident with the peak of AP expression in differentiation culture conditions (days 4-6), and S3, corresponding to the onset of mineral deposition in the extracellular matrix (days 7-9). The results showed that conditioned medium of ROB contains greater amounts of IGF-II than IGF-I at all differentiation stages. Both peptides showed the highest concentrations on day 3 of differentiation (end of S1). All IGF-binding proteins (IGFBPs), except IGFBP-1 and -6, were detected, and IGFBP-2 was the most abundant IGFBP present in the conditioned media, and its degradation increased from S1 to S3. By semiquantitative RT-PCR, IGF-I and IGF-II were highly expressed on days 3 and 6, whereas IGFBP-2 was constantly expressed. We focused our study on the role of IGF-II and IGFBP-2 on the synthesis of AP, an early marker of osteoblast maturation. The results showed that a significant increase in AP expression was induced by IGF-II added to the differentiating osteoblasts continuously or in S1 but not in S2 or S3. IGFBP-2 was able to potentiate endogenous and exogenous IGF-II-dependent stimulation of AP activity, and its proteolytic degradation in late stages of osteogenesis (S2 and S3) was highly correlated with the increase of active matrix metalloproteinase-2 in the CM and with the decreased efficacy of IGF-II action. These data suggest that IGFBP-2, at nearly equimolar concentration with IGF-II, plays a potentiating role in IGF-II action on ROB differentiation in vitro.