A key impediment to the development of effective virus-mediated gene therapy for cancer is the low level of gene transfer that occurs after the administration of recombinant viral vectors. Improving in vivo infection and transduction efficiency is an important goal for gene therapy. The limited distribution of gene delivery is particularly problematic when large vectors such as recombinant adenoviruses and retroviruses are used to mediate transgene delivery to solid tumors. To facilitate the spread of virus, we have investigated the potential of administering proteases prior to the intratumoral inoculation of recombinant replication deficient adenovirus. For these studies, we chose proteases that are active against collagen and the other extracellular matrix proteins found in primary brain tumor tissue, but are not widely expressed in normal brain. Various concentrations of a mixture of collagenase/dispase or trypsin were inoculated into xenografts of human glioblastoma multiforme-derived brain tumor cell lines U87, U251, and SF767. Subsequently, recombinant adenovirus encoding the beta-galactosidase gene was administered and tumor tissue was examined for evidence of virus infection. Both collagenase/dispase and trypsin enhanced virus infection, indicating that protease pretreatment may be a useful strategy for enhancing virus-mediated gene transduction for many in vivo applications.