We previously identified and characterized amino acid substitutions in a loop connecting helix I to strand B, the aI/pB loop, of the N-domain that are critical for in vivo folding of the maltose-binding protein (MalE31). The tertiary context-dependence of this mutation in MalE folding was assessed by probing the tolerance of an equivalent ap loop of the C-domain to the same amino acid substitutions (MaIE219). Moving the loop mutation from the N-to the C-domain eliminated the in vivo misfolding step that led to the formation of inclusion bodies. In vitro, both loop variants exhibited an important decrease of stability, but their intrinsic tendency to aggregate was well correlated with their periplasmic fates in Escherichia coli. Furthermore, the noncoincidence of the unfolding and refolding transition curves and increase of light scattering during the refolding of MalE31 indicate that a competing off-pathway reaction could occurs on the folding pathway of this variant. These results strongly support the notion that the formation of supersecondary structures of the N-domain is a rate-limiting step in the folding pathway of MalE.