Skeletal muscle is an attractive target for somatic gene transfer of both acquired and inherited disorders. Direct injection of adenoviral vectors in the skeletal muscle leads to recombinant gene expression in a large number of muscle fibers. Transgene expression has been transient in most organs and associated with substantial inflammation when experiments are performed in adult immune competent mice. In this report, we utilize a variety of in vivo and in vitro models of T and B cell function to characterize the nature of the immune response to adenoviral vectors injected into murine skeletal muscle. Cellular immunity dependent on CD4+ and CD8+ T cells contributes to the loss of recombinant gene expression and the development of localized inflammation. Antigen specific activation of T cells occurs to both viral proteins and the reporter gene beta-galactosidase. Systemic levels of neutralizing antibody to the capsid proteins of the vector are also generated. Destructive immune responses responsible for loss of transgene expression are largely directed against beta-galactosidase in that transgene expression was stable when beta-galactosidase was eliminated as a neoantigen in mice transgenic for lacZ. A strategy to prevent the cellular and humoral immunity to this therapy was developed based on transiently ablating CD4+ T cell activation at the time of vector delivery. Encouraging results were obtained when vector was administered with one of several immune modulating agents including cyclophosphamide, mAb to CD4+ cells, and mAb to CD40 ligand. These studies indicate that cellular and humoral immune responses are elicited in the context of gene therapy directed to skeletal muscle with adenoviral vectors. Transient ablation of CD4+ T cell activation prevents the effects responses of the CD8+ T and B cells.