Objective: Human insulin-like growth factor-I and -II (IGF-I and -II) ligands share a high degree of sequence and structural homology. Despite their similarities, ICF-I and IGF-II exhibit differential receptor binding and activation characteristics. The C domains of IGF-I and IGF-II are the primary determinants of binding specificity to the insulin-like growth factor I receptor (IGF-IR), insulin receptor exon 11 - (IR-A) and exon 11 + (IR-B) isoforms. Design: Three IGF-II analogues were generated in order to delineate the C domain residues that confer the differential receptor binding affinity and activation properties of the IGFs. Chimeric IGF-II analogues IGF-IICIN and IGF-IICIc contained partial IGF-I C domain substitutions (IGF-I residues underlined) GYGSSSRRSR and SRVSRRAPQT, respectively. Results: The IGF-IICIN analogue bound the IR-A and IGF-IR with high affinity but bound the IR-B with a relatively lower affinity than IGF-II, suggesting a negative interaction between the exon-11 encoded peptide in the IR-B and the C-domain. The ability of IGF-IICIN to activate receptors and elicit cell viability responses was generally proportional to its relative receptor binding affinity but appeared to act as a partial agonist equivalent to IGF-I when binding and activating the IGF-IR. In contrast. IGF-IICIc bound IGF-IR with high affinity but elicited lower receptor activation and cell viability responses. Analogue IGF-IICIs contained a truncated IGF-I C domain (GSSSRRAT) and generally displayed a relatively poor ability to bind, activate and elicit viability responses via each receptor. Conclusions: Together, the IGF analogues demonstrate that both flanks of the IGF-IIC domain play important roles in the greater ability of IGF-II to bind and activate IR receptors than IGF-I.