A cDNA for a human interleukin-3 (hIL-3) binding protein has been isolated by a novel expression cloning strategy: a cDNA library was coexpressed with the cDNA for the beta subunit of human granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor (hGMR beta) in COS7 cells and screened by binding of 125I-labeled IL-3. The cloned cDNA (DUK-1) encodes a mature protein of 70 kd, which belongs to the cytokine receptor family and which alone binds hIL-3 with extremely low affinity (Kd = 120 +/- 60 nM). A high affinity IL-3-binding site (Kd = 140 +/- 30 pM) was reconstituted by coexpressing the DUK-1 protein and hGMR beta, indicating that hIL-3R and hGMR share the beta subunit. Therefore, we designated DUK-1 as the alpha subunit of the hIL-3R. As in human hematopoietic cells, hIL-3 and hGM-CSF complete for binding in fibroblasts expressing the cDNAs for hIL-3R alpha, GMR alpha, and the common beta subunit, indicating that different alpha subunits compete for a common beta subunit.