Insulin-like growth factor 1 and 2 (IGF1 and -2) are potent stimulators of diverse cellular activities such as differentiation and mitosis. Six IGF binding proteins (IGFBP1 through -6) are primary regulators of IGF half-life and receptor availability. Generally, the binding of IGFBPs inhibits IGF-receptor activation. However, it has been shown that IGFBP2 in complex with IGF2 (IGF2/IGFBP2) stimulates osteoblast function in vitro and increases skeletal mass in vivo. IGF2 binding to IGFBP2 greatly increases the affinity for 2- or 3-carbon O-sulphated glycosaminoglycans (GAGs), e.g., heparin and heparan sulphate, which is hypothesized to preferentially and specifically target the IGF2/IGFBP2 to the bone matrix. In order to obtain a more detailed understanding of the interactions between IGF2/IGFBP2 and GAGs, we investigated heparin binding properties of IGFBP2 and the IGF2/IGFBP2 in a quantitative manner. For this study, we mutated key positively charged residues within the two heparin binding domains (HBDs) in IGFBP2 and in one potential HBD in IGF2. Using heparin affinity chromatography, we demonstrate that the two IGFBP2 HBDs contribute differentially to GAG binding in the free IGFBP2 and the IGF2/IGFBP2 protein complex. Moreover, we identify a significant contribution from the HBD in IGF2 to the increased IGF2/IGFBP2 heparin affinity. Using molecular modeling, we present a novel model for the IGF2/IGFBP2 interaction with heparin where all three proposed HBDs constitute a positively charged and surface-exposed area that would serve to promote the increased heparin affinity of the complex compared to free intact IGFBP2.