Adenoviral (Ad) vectors demonstrate several attributes of potential utility for glioma gene therapy. Although Ad infection is limited in vitro by low expression levels of the coxsackie-adenoviral receptor (CAR), in vivo studies have shown the efficacy of Ad vectors as gene delivery vectors. To evaluate the in vivo utility of CAR-independent, infectivity-enhanced Ad vectors, we employed genetically modified Ad vectors in several experimental models of human gliomas. We used three capsid-modified Ad vectors: (1) a chimeric Ad vector with a human Ad backbone and a fiber knob of a canine Ad, (2) an Ad vector with a polylysine motif incorporated into the fiber gene, and (3) a double-modified Ad vector incorporating both an RGD4C peptide and the polylysine motif. These three modified Ad vectors target, respectively, the putative membrane receptor(s) of the canine Ad vector, heparan sulfate proteoglycans (HSPGs), and both integrins and HSPGs. Our in vitro studies indicated that these retargeting strategies all enhanced CAR-independent infectivity in both established and primary low-passage glioma cells. Enhancement of in vitro gene delivery by the capsid-modified vectors correlated inversely with the levels of cellular CAR expression. However, in vivo in orthotopic human glioma xenografts, the unmodified Ad vector was not inferior relative to the capsid-modified Ad vector. Although genetic strategies to circumvent CAR deficiency in glioma cells could reproducibly expand the cellular entry mechanisms of Ad vectors in cultured and primary glioma cells, these approaches were insufficient to confer in vivo significant infectivity enhancement over unmodified Ad vectors. Other factors, probably the extracellular matrix, stromal cells, and the three-dimensional tumor architecture, clearly play important roles in vivo and interfere with Ad-based gene delivery into glioma tumors.