Adenovirus is a significant pathogen in immunocompromised patients and is widely utilized as a gene delivery vector, so a detailed understanding of the human immune response to adenovirus infection is critical. This study characterized the adenovirus-specific CD4(+) T-cell response of healthy donors by incubation with whole virus or with individual hexon and fiber proteins. Adenovirus-specific CD4(+) T cells averaged 0.26 % of the CD4(+) T-cell pool and were detectable in all donors. T cells recognizing the highly conserved hexon protein accounted for 0.09 %, whereas no response was observed against the fiber protein. A panel of hexon-specific CD4(+) T-cell clones was generated and shown to lyse targets infected with adenovirus from different serotypes and species. Three CD4 T-cell epitopes are described, which map to highly conserved regions of the hexon protein.