Leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), and oncostatin M (OSM) are four helix bundle cytokines acting through a common heterodimeric receptor composed of gp130 and LIF receptor (LIFR). Binding to LIFR occurs through a binding site characterized by an FXXK motif located at the N terminus of helix D (site III). The immunoglobulin (Ig)-like domain of LIFR was modeled, and the physico-chemical properties of its Connolly surface were analyzed. This analysis revealed an area displaying properties complementary to those of the LIF site III. Two residues of the Ig-like domain of LIFR, Asp214 and Phe284, formed a mirror image of the FXXK motif. Engineered LIFR mutants in which either or both of these two residues were mutated to alanine were transfected in Ba/F3 cells already containing gp130. The F284A mutation impaired the biological response induced by LIF and CT-1, whereas the response to OSM remained unchanged. The Asp214 mutation did not alter the functional responses. The D214A/F284A double mutation, however, totally impaired cellular proliferation to LIF and CT-1 and partially impaired OSM-induced proliferation with a 20-fold increase in EC50. These results were corroborated by the analysis of STAT3 phosphorylation and Scatchard analysis of cytokine binding to Ba/F3 cells. Molecular modeling of the complex of LIF with the Ig-like domain of LIFR provides a clue for the superadditivity of the D214A/F284A double mutation. Our results indicate that LIF, CT-1, and OSM share an overlapping binding site located in the Ig-like domain of LIFR. The different behaviors of LIF and CT-1, on one side, and of OSM, on the other side, can be related to the different affinity of their site III for LIFR.