The growth plate is an important target tissue for insulin-like growth factors (IGFs), but little is known on the regulation of the IGF system during the developmental sequence of chondrocytes. We therefore examined the expression profile of IGF system components in proliferating vs. differentiating growth plate chondrocytes by use of two cell culture models of the growth cartilage. In rat growth plate chondrocytes in primary culture, IGF-I expression increased 2-fold during the process of differentiation. IGFBP-3 expression showed a biphasic pattern of with a 2-fold increase at the onset of differentiation and a down-regulation in late differentiating chondrocytes to 25% of baseline levels; the expression pattern of IGFBP-2, IGFBP-4 and IGFBP-6 was not dependent on the developmental stage. In IGF- and IGFBP-3-deficient RCJ3.1C5.18 (RCJ) mesenchymal chondrogenic cells, IGFBP-2- and IGFBP-6 synthesis declined by 50% during differentiation. IGFBP-5 expression was markedly upregulated during the process of differentiation in both cell culture models. While IGFBP-5 overexpression did not have an IGF-independent effect on RCJ cell differentiation, it promoted IGF-I-enhanced differentiation of these cells. A potential mechanism for this effect is the specific increase of Akt phosphorylation in IGFBP-5-overexpressing cells in the presence of IGF-I, indicating an increased activity of the phosphatidylinositol-3 kinase pathway. These data suggest that the developmental stage of the chondrocyte is an important determinant of IGF and IGFBP expression and imply a functional role for IGFBP-5 for upregulating IGF action during chondrocyte differentiation in vivo.