Although cellular immune responses are likely important for recovery from acute adenovirus infection, they have not been studied in humans. As a first step, a sensitive assay for the detection of adenovirus-specific proliferative T cell responses was developed. Peripheral blood mononuclear cells from 29 of 30 healthy adults exhibited specific proliferative responses to adenovirus type 2 antigen. Adenovirus-specific proliferation was found to be mediated by CD4+ T cells. Specific proliferative responses were detected to purified Ad2 virions and by Ad2-infected cell lysates, indicating that adenovirus structural proteins are important targets. In addition, specific proliferative responses to the uncommon adenovirus type 35 were found in persons without serologic evidence of prior Ad35 infection. These data suggest that adenovirus-specific CD4+ T cells recognize conserved antigens among different serotypes and that a majority of persons develop long-lived CD4+ T cell responses to adenovirus.