The mammalian insulin-like growth factor (IGF)-II/cation-independent mannose 6-phosphate receptor (IGF2R) binds IGF-II with high affinity. By targeting IGF-II to lysosomal degradation, it plays a role in the maintenance of correct IGF-II levels in the circulation and in target tissues. Loss of IGF2R function is associated with tumor progression; therefore, the IGF2R is often referred to as a tumor suppressor. The interaction between IGF2R and IGF-II involves domains 11 and 13 of the 15 extracellular domains of the receptor. Recently, a hydrophobic binding region was identified on domain 11 of the IGF2R. In contrast, relatively little is known about the residues of IGF-II that are involved in IGF2R binding and the determinants of IGF2R specificity for IGF-II over the structurally related IGF-I. Using a series of novel IGF-II analogues and surface plasmon resonance assays, this study revealed a novel binding surface on IGF-II critical for IGF2R binding. The hydrophobic residues Phe19 and Leu53 are critical for IGF2R binding, as are residues Thr16 and Asp52. Furthermore, Thr16 was identified as playing a major role in determining why IGF-II, but not IGF-I, binds with high affinity to the IGF2R. ; Carlie Delaine, Clair L. Alvino, Kerrie A. McNeil, Terrance D. Mulhern, Lisbeth Gauguin, Pierre De Meyts, E. Yvonne Jones, James Brown, John C. Wallace, and Briony E. Forbes