The crucial step of folding of recombinant proteins presents serious challenges to obtaining the native structure. This problem is exemplified by insulin-like growth factor (IGF)-I which when refolded in vitro produces the native three-disulfide structure, an alternative structure with mispaired disulfide bonds and other isomeric forms. To investigate this phenomenon we have examined the refolding properties of an analog of IGF-I which contains a 13-amino acid N-terminal extension and a charge mutation at position 3 (Long-[Arg 3 ]IGF-I). Unlike IGF-I, which yields 45% of the native structure and 24% of the alternative structure when refolded in vitro, Long-[Arg 3 ]IGF-I yields 85% and 10% of these respective forms. To investigate the interactions that affect the refolding of Long-[Arg 3 ]IGF-I and IGF-I, we acid-trapped folding intermediates and products for inclusion in a kinetic analysis of refolding. In addition to non-native intermediates, three native-like intermediates were identified, that appear to have a major role in the in vitro refolding pathway of Long-[Arg 3 ]IGF-I; a single-disulfide Cys 18 -Cys 61 intermediate, an intermediate with Cys 18 -Cys 61 and Cys 6 -Cys 48 disulfide bonds and another with Cys 18 -Cys 61 and Cys 47 -Cys 52 disulfide bonds. Furthermore, from our kinetic analysis we propose that the Cys 18 -Cys 61 , Cys 6 -Cys 48 intermediate forms the native structure, not by the direct formation of the last (Cys 47 -Cys 52 ) disulfide bond, but by rearrangement via the Cys 18 -Cys 61 intermediate and a productive Cys 18 -Cys 61 , Cys 47 -Cys 52 intermediate. In this pathway, the last disulfide bond to form involves Cys 6 and Cys 48 . Finally, we apply this pathway to IGF-I and conclude that the divergence in the in vitro folding pathway of IGF-I is caused by non-native interactions involving Glu 3 that stabilize the alternative structure.