Summary The carboxyl(C)-terminus of human interleukin-6 (hIL-6) has a critical role in the expression of the biological activity of this cytokine. To define the structure-function relationships of this region, semi-random mutagenesis of the C-terminal Leu181-Arg182-Qln183-Met184 sequence of hIL-6 was performed. The mutants were produced in Escherichia coli, renatured, and purified. Alterations of the C-terminal 4 amino acids caused a significant reduction of the proliferative effect of the mutants on MH60.BSF2 and KT-3 cells, and also led to a drastic decrease in receptor binding affinity. These results suggest the importance of a positively charged residue at position 182 or 183 and an alpha-helix at position 181 for the biological activity of hIL-6.