This study was designed to examine the potential for gene therapy in bladder in vivo using adenoviral vectors. Gene transfer to rat bladders was accomplished via direct intravesical instillation using a replication-defective adenoviral vector containing a marker gene encoding for Escherichia coli beta-galactosidase (beta-gal). Successful gene transfer was confirmed by analyzing bladder samples for DNA and RNA using polymerase chain reaction (PCR) with primers specific for beta-gal and adeno sequences, detecting beta-gal in full-thickness bladder wall using specific histochemical staining (X-gal) and documenting recombinant protein production. Bladder architecture was preserved, without evidence of distant spread of virus as assessed by PCR. Gene expression was evident for at least 7 days. In summary, bladder cells can be genetically altered using replication-deficient adenoviral vectors via simple intravesical instillation of vector. Introduction of exogenous genetic material is a potentially powerful therapeutic modality for immunomodulation of bladder neoplasms.