The adenoviruses are nonenveloped double stranded DNA viruses, which cause enteric dysentery and respiratory infection. Adenovirus has become a focus of the water treatment community because of its apparent resistance to ultraviolet disinfection; it is the basis for stringent new EPA regulations regarding UV disinfection of all viruses in both surface and ground waters. Most of the data generated for disinfection studies has involved the use of monochromatic (254 nm) low pressure (LP) UV sources and subsequent assay of viral infectivity in cell culture models. LP UV lamps primarily damage DNA, while polychromatic UV sources may damage other parts of the virus as well. Recent research has shown that these newer, polychromatic UV sources—such as medium pressure (MP) UV—are more effective than monochromatic LP UV for disinfection of adenovirus; however, the reasons for this increased effectiveness are not known. The objectives of this work were to study adenoviral response to UV using both LP and MP UV as well as using both standard cell culture infectivity assays and more direct methods of assessment based on molecular biology. These include quantitative long PCR for assessment of DNA damage and SDS-PAGE for assessment of protein damage; transmission electron microscopy was used to examine the structure of UV treated viral particles. This work was only the second significant study to show the response of adenoviruses to medium pressure UV and the first to thoroughly examine the response of adenoviruses to both LP and MP UV using cell culture-independent methods. Results confirm that adenovirus is sensitive to MP UV when assayed in cell culture; they show that LP and MP UV are equally effective at inducing damage to the adenoviral genome and that MP UV is more effective than LP UV at damaging the viral proteins. This work helps deepen our understanding of UV disinfection of adenovirus.