Recombinant adenoviruses (Ads) are useful tools in gene transfer because they are able to infect a wide variety of tissues and cell types and do not require a replicating target cell. However, transgene expression is only transient due to host innate and acquired immune responses to the virus. Most recombinant Ads have deletions of early region 3 (E3) genes, allowing more space for insertion of the transgene. Although the E3 region is not necessary for infection, it has been observed that these "nonessential" genes have immunomodulatory properties. We demonstrate here that the E3 region of Ad inhibits the activation of NF-kappa B induced by tumor necrosis factor alpha (TNF-alpha) and interleukin-1. Ad E3 is able to prevent NF-kappa B from entering the nucleus, where it is normally active. Ad E3 also appears to function by preventing the activation of the kinase complex, IKK, which is responsible for phosphorylation of I kappa B that retains NF-kappa B in the cytoplasm in an inactive state. The prevention of NF-kappa B activation has been mapped to a complex of two of the seven E3 products, E3-10.4K and E3-14.5K (RID alpha/beta). These and other studies indicate that, by using Ad vectors containing the E3 region, it may be possible to reduce the harmful proinflammatory effects of TNF-alpha and other cytokines that compromise the use of Ad gene therapy vectors in vivo.