We have identified functionally important regions of human interleukin-11 (hIL-11) by means of alanine-scanning mutagenesis. A total of 61 mutated forms of hIL-11 were produced in E. coli as thioredoxin fusion proteins and tested in a murine T10 plasmacytoma proliferation assay. Mutations made at several positions proximal to the hIL-11 C-terminus caused substantial reduction in biological activity. In addition a number of other mutations in this region affected either protein folding or stability. Both effects displayed a characteristic periodicity with respect to the primary sequence which suggested that residues close to the C-terminus of hIL-11 adopt a helical conformation. Mutations made proximal to the N-terminus of hIL-11 also exhibited reduced bioactivity, although no effects on protein folding or stability were observed. The N-terminal mutations with reduced activity also mapped with a periodicity suggestive of a helical conformation. We previously have proposed a four-helix bundle topology for the hIL-11 structure based on physical studies, selective chemical modifications, positions of intron/exon boundaries, limit proteolysis experiments and site-directed mutagenesis. The alanine-scanning mutagenesis data we report here provide additional support for this model.