Sequence randomization through functional phage display of single chain human interleukin (IL)-5 was used to investigate the limits of replaceability of the Glu110 residues that form a part of the receptor-binding epitope. Mutational analysis revealed unexpected affinity for IL-5 receptor α chain with variants containing E110W or E110Y. Escherichia coli-expressed Glu110 variants containing E110W in the otherwise sequence-intact N-terminal half, including a variant with an E110A replacement in the sequence-disabled C- terminal half, were shown by their CD spectra to be folded into secondary structures similar to that of single chain human IL-5 (scIL-5). Biosensor kinetics analysis revealed that (E110W/A5)scIL-5 and (E110W/A6)scIL-5 had receptor α chain binding affinities similar to that of (wt/A5)scIL-5. However, (E110W/A6)scIL-5 had a significantly reduced bioactivity in TF-1 cell proliferation compared with both (wt/A5)scIL-5 and (E110W/A5)scIL-5, and this activity reduction was disproportionately greater than the much smaller effect of Glu110 mutation on receptor binding affinity. The marked and disproportionate decrease in TF-1 proliferation observed with (E110W/A6)scIL- 5 suggests a role for Glu110 in the biological activity mediated by the signal transducing receptor βc subunit of the IL-5 receptor. This is also consistent with the lack of stimulation of JAK2 phosphorylation by the (E110W/A6)scIL-5 mutant in recombinant 293T cells, as compared with the concentration-dependent stimulation seen for scIL-5. The results reveal the dispensability of charge in the Glu110 locus of IL-5 for receptor α chain binding and, in contrast, its heretofore underappreciated importance for receptor activation.