A family of six insulin-like growth factor (IGF) binding proteins (IGFBP-1-6) binds IGF-I and IGF-II with high affinity and thus regulates their bioavailability and biological functions. lGFBPs consist of N- and C-terminal domains, which are highly conserved and cysteine-rich, joined by a variable linker domain. The role of the C-domain in IGF binding is not completely understood in that C-domain fragments have very low or even undetectable IGF binding affinity, but loss of the C-domain dramatically disrupts IGF binding by IGFBPs. We recently reported the solution structure and backbone dynamics of the C-domain of IGFBP-2 (C-BP-2) and identified a pH-dependent heparin binding site [Kuang, Z., Yao, S., Keizer, D. W., Wang, C. C., Bach, L. A., Forbes, B. E., Wallace,.J. C., and Norton, R. S. (2006) Structure, dynamics and heparin binding of the C-terminal domain of insulin-like growth factor-binding protein-2 (IGFBP-2), J. Mol. Biol. 364, 690-704]. Here, we have analyzed the molecular interactions among the N-domain of IGFBP-2 (N-BP-2), C-BP-2, and IGFs using cross-linking and nuclear magnetic resonance (NMR) spectroscopy. The binding of C-BP-2 to the IGF-I-N-BP-2 binary complex was significantly stronger than the binding of C-BP-2 to IGF-I alone, switching from intermediate exchange to slow exchange on the NMR time scale. A conformational change or stabilization of the IGF-I Phe49-Leu54 region and the Phe49 aromatic ring upon binding to the N-domains, as well as an interdomain C interaction between N-BP-2 and C-BP-2 (which is also detectable in the absence of ligand), may contribute to this cooperativity in IGF binding. Glycosaminoglycan binding by IGFBPs can affect their IGF binding although the effects appear to differ among different IGFBPs; here, we found that heparin bound to the IGF-I center dot N-BP-2 center dot C-BP-2 ternary complex, but did not cause it to dissociate.