We have developed a humanized, bispecific immunoadhesin-antibody (BsIAb) that targets and kills HIV-infected cells. Comprised of CD4-IgG and humanized anti-CD3-IgG, this BsIAb is bifunctional. First, in targeting, it exploits the natural affinity of CD4 for gp120 to target the BsIAb to HIV-infected cells, and second, it recruits and activates, through its anti-CD3 moiety, cytotoxic T lymphocytes (CTL) to lyse target cells in a non-MHC restricted manner. To produce purified BsIAb from supernantants of transfected mammalian cells, we designed a three-step recovery scheme based on the structural elements of this heterotrimeric protein. The ability of purified BsIAb to specifically lyse HIV-infected target cells was demonstrated using CTL from two different sources: whole peripheral blood lymphocyte (PBL) fractions and pure CTL preparations. In contrast, a human anti-gp120 antibody mediated lysis of HIV-infected target cells only with PBL fractions and not with purified CTL. Moreover, lysis observed in the presence of the human anti-gp120 antibody was completely blocked in the presence of human serum (which competes for Fc gamma receptor binding), whereas BsIAb-mediated lysis of target cells was not affected. We measured the monovalent affinities of BsIAb for HIV-gp120 on infected cells and for CD3 epsilon on CTL. Relative to the bivalent parent molecules, CD4/gp120 affinity in the BsIAb is unchanged, whereas anti-CD3/CD3 is substantially decreased. We further demonstrated by fluorescence microscopy that physical association of CD3+ cells with gp120-expressing cells occurs only in the presence of BsIAb. Thus, the cytocidal activity of BsIAb in the presence of serum reflects its unique ability to recruit CTL as effector cells and highlights a potentially important advantage of this type of construct over antibodies for HIV-directed therapy.