Efficient gene transfer by recombinant adenovirus (Ad) vectors depends on expression of CAR and α(v) integrin on target cells. Because Ad may also infect nearby nontarget cells expressing these receptors, such as peritoneal mesothelial cells after i.p. injection, we hypothesized that targeting Ad gene delivery to a receptor overexpressed on most ovarian carcinoma cells, such as TAG-72, would enhance the selectivity of Ad gene transfer when used in this context. A monoclonal antibody that has been investigated clinically for immunotherapy and immunodetection of ovarian carcinomas, namely CC49, was used to construct a bispecific conjugate with the Fab fragment of a neutralizing anti-knob mAb to target Ad binding via TAG-72. This conjugate facilitated TAG-72-specific, CAR-independent Ad reporter gene transfer to both ovarian cancer cell lines and primary ovarian cancer cells cultured from malignant ascites fluid. Fab-CC49 was very selective for tumor cells, augmenting Ad gene transfer to primary ovarian cancer cells 2- to 28-fold relative to untargeted Ad, while also decreasing gene transfer to autologous cultured mesothelial cells 4- to 9-fold. These data suggest that targeting Ad via TAG-72 may improve the selectivity of Ad gene transfer for ovarian tumors 8- to 252-fold on i.p. vector injection. These results also define the requirements for a candidate target receptor in the rational design of a targeted Ad vector for ultimate clinical utility, one that selectively infects tumor cells and spares normal cells on i.p. injection. Such a vector may increase gene transfer and decrease the toxicity of Ad vectors, which would improve the therapeutic index of cytotoxic gene therapy for ovarian cancer in clinical trials.