The use of shRNA for knockdown of gene expression is a powerful method. In addition to transient transfection of RNA oligonucleotides, various DNA-based vectors that express short hairpin RNAs have been successfully used for efficient depletion of gene products. Replication-defective retrovirus and adenovirus (Ad) vectors have also gained wide usage. The extension of shRNA technology to replication-competent Ad would be desirable to investigate the role of various cellular genes in Ad replication. This approach is hampered because the effect of shRNA is neutralized by the Ad VA-RNA that is expressed at late stages after infection, and the infected cells are killed prior to significant depletion of some long-lived target gene products. We have constructed replication-competent Ad vectors for the depletion of the pro-apoptotic proteins BAX and BAK. We have modified a replication-defective Ad multivalent shRNA expression vector developed by Welgen, Inc. In our vector design, the multivalent shRNA expression cassette is contained in the E1B region. Additionally, we have incorporated a temperature-sensitive mutation in the viral DBP gene (ts125). The use of this vector has resulted in efficient depletion of critical cellular apoptotic modulators, BAX and BAK. This vector may be useful to study the role of various cellular genes in Ad-induced apoptosis and viral replication.