Nonsensitized human and mouse lymphocytes produce interferon (IFN)-α and IFN-α/β, respectively, in response to most transformed cells, as well as normal xenogeneic cells. The treatment of transformed human (WISH) and mouse (L) or normal mouse embryo (ME) cells with trypsin, pepsin, or neuraminidase resulted in a loss of the cell's ability to induce IFN-α or α/β. These findings suggested that a cell surface glycoprotein is responsible for the induction of IFN-α and α/β. The sonication of WISH, L, and ME cells released glycoproteins which retained IFN-inducing activity in the soluble form. The inducing molecules from WISH, L, and ME cells had molecular weights of 130,000, 90,000, and 68,000, respectively. Further purification, plus a confirmation of the glycoprotein nature of the inducer molecule, was shown by specifically binding and eluting L cell inducer bioactivity from a concanavalin A-Sepharose affinity column.