Thrombopoietin (TPO) is a haematopoietic growth factor responsible for megakaryocyte progenitor proliferation and differentiation. It belongs to the four-helix-bundle cytokine family and exerts its biological effects through binding to a specific receptor, c-Mpl. With the use of site-directed mutagenesis we have generated 20 TPO mutants. Each of the TPO mutants was produced in a eukaryotic expression system and the mutants' ability to induce the proliferation of factor-dependent c-Mpl-expressing megakaryoblastic M-O7e cells was compared with that of wild-type TPO. Among the mutations studied, 10 lead to a significant decrease in TPO bioactivity. Of these ten residues, three are located in helix A of the protein (Arg¹⁰, Lys¹⁴ and Arg¹⁷) and four in helix D (His¹³³, Gln¹³², Lys¹³⁸ and Phe¹⁴¹), indicating that in TPO, as in other cytokines, these two helices are important for functional cytokine/receptor interactions. Surprisingly, mutant Arg¹⁰ → Ala (R10A) lacked any proliferative activity, despite the fact that this mutation was recently reported to have no effect on TPO/c-Mpl binding in a TPO phage ELISA [Pearce, Potts, Presta, Bald, Fendly and Wells (1997) J. Biol. Chem. 272, 20595–20602]. The lack of M-O7e proliferation is probably due to an inability of R10A mutant to promote receptor dimerization and thus receptor activation. Moreover we found that the Arg¹⁰ and Arg¹⁷ residues of TPO seem to be specific determinants for TPO/c-Mpl recognition. We also demonstrate that the O-glycosylation site located at position 110 of TPO is not necessary for the bioactivity of the cytokine.