The effectiveness of cellular immunotherapy of solid tumors is often hampered by the lack of specific infiltration of immune effector cells into the tumor mass. Therefore, we studied the potential of tumor antigen-specific antibodies to elicit tumor-specific myeloid cell activation, to induce or enhance tumor infiltration by immune cells. To this end, we developed an in vitro model system using the human myeloid cell line MonoMac-6. Incubation of IFN-gamma-primed MonoMac-6 cells with serum-opsonized zymosan or EGP-2-directed, mouse IgG2a-opsonized, EGP-2-positive tumor cells resulted in the production of ROS and TNF-alpha and induced E-selectin and ICAM-1 expression on HUVECs. FcR-mediated MonoMac-6 cell activation was strictly dependent on the activation of MonoMac-6 cells with IFN-gamma. In addition, no myeloid cell activation was observed in the presence of human serum or using tumor antigen-specific mouse antibody subclasses other than IgG2a, suggesting the crucial involvement of CD64 (FcgammaR1) in the effects observed. However, serum-inhibited myeloid cell activation was completely restored employing a 2-step targeting approach in which tumor cell opsonization with mouse anti-EGP-2 antibodies was followed by incubation with human antimouse Ig antibodies. Moreover, using this 2-step approach, not only anti-EGP-2-directed mouse IgG2a but also mouse IgG1 antibodies effectively induced tumor-specific myeloid cell activation. In conclusion, we describe a method to induce efficient and tumor-specific activation of myeloid cells based on the sequential use of mouse tumor antigen-specific and human antimouse Ig antibodies. Targeted myeloid cell activation may provide a means to aid in the induction of a tumor-directed immune response and as such, the method described here could be of clinical significance.