Adenoviral-mediated gene transfer is suboptimal in human glioma and limits in vivo gene therapy approaches. There is a need for targeted vectors able to enhance gene transfer into the tumor as well as to lower the viral load in the surrounding normal tissues. We evaluated primary human tumor samples by immunohistochemistry and fluorescence-activated cell sorter for expression of the Coxsackie-adenovirus receptor and other antigens with potential utility to redirect adenoviruses (Ads) to gliomas. In the majority of the samples, Coxsackie-adenovirus receptor expression was low. This correlated with inefficient gene transfer in vitro. Epidermal growth factor receptor (EGFR) and alpha(v)beta5 integrins were often highly, but heterogeneously, expressed. We hypothesized that these receptors, overexpressed in tumor but not in normal brain, could serve as independent binding sites for alternative pathways of infection with targeted Ads. We examined this, using Ads that expressed the luciferase reporter gene under the cytomegalovirus promoter. Targeting to the EGFR was performed with a single-chain bispecific antibody directed against the human EGFR and against the fiber knob of the Ad. Targeting to the alpha(v) integrins was performed by insertion of an integrin-binding sequence, RGD-4C, in the HI-loop of the Ad. Increased luciferase gene transfer in primary glioma cells was observed in 8 of 13 samples with EGFR-targeting (2-11 times enhancement; median, 6) and in all of the samples with RGD-targeting (2-42 times enhancement; median, 12). Combining the two targeting motifs further enhanced the gene transfer in primary glioma cells in an additive manner (3-56 times; median, 20). The double-targeted Ads also strongly augmented gene transfer into organotypic glioma spheroids. Conversely, gene transfer into normal brain explants was reduced dramatically using Ads targeted to the tumor. Our findings demonstrate the feasibility and benefit of binding multiple ligands to the adenoviral fiber knob. These vectors have a great potential for clinical use in the context of tumors that are usually heterogeneous for target antigen expression at the single-cell level.