Bispecific antibodies (bsAb) have attracted much attention over the past several years as a mean to improve immunotherapy of cancer. Due to their dual specificity, bsAb are able to redirect effector cells against tumor targets. In this study, the development and preclinical testing of a new quadroma-derived bsAb, HEA125x197, recognizing the tumor-associated Ep-CAM antigen and the high affinity Fc receptor for IgG, CD64, is reported. Using granulocyte-colony stimulating factor (G-CSF) and interferon-gamma (IFN-gamma)-stimulated polymorphonuclear neutrophils to induce CD64 expression, bsAb HEA125 x 197 elicited strong cytotoxic activity towards allogeneic and autologous ovarian carcinoma cells. The cytolytic efficiency of this antibody was comparable to that of a previously described bsAb, HEA125 x OKT3, targeting preactivated T lymphocytes against Ep-CAM-carrying tumor cells. Based on the pan-carcinoma specificity and the stable expression of Ep-CAM, bsAb HEA125x197 may broaden the spectrum of bispecific reagents for the treatment of epithelial malignancies.