Blood-cell production is regulated by hemopoietic growth factors, which act at specific stages of hemopoietic cell differentiation. Murine interleukin-3 (mIL-3)/multilineage colony-stimulating factor (multi-CSF) has been shown to stimulate colony formation in vitro by multipotent hemopoietic cells and production of spleen colony-forming units (CFU-S) in suspension cultures. The molecular cloning of the human counterpart of mIL-3 is described here. Hybridization of radiolabeled mIL-3 cDNA with a cDNA library obtained from mRNA of stimulated human lymphocytes resulted in the identification of a human (h)multi-CSF cDNA clone. Sequence homology (73%) in the 3'-noncoding region of mIL-3 enabled the detection of the hmulti-CSF cDNA clone. Whereas only 45% sequence homology was found in the coding region, specific A + T-rich domains in the 3'-noncoding region were highly conserved (93%). As far as we know, this is the first example of gene identification by sequence homology occurring only within the 3'-noncoding region. The protein encoded by this hmulti-CSF cDNA stimulates in vitro colony formation by multipotent human hemopoietic stem cells. In addition, the growth factor strongly stimulates the in vitro proliferation of human leukemic blast cells.