For cancer gene therapy, a recombinant adenovirus serotype 5 named RPR/INGN201 has been constructed by susbtitution of the E1 region with human tumor suppressor gene p53. The protein components of RPR/INGN201 virions were separated by reversed-phase HPLC and were individually identified by electrospray time-of-flight mass spectrometry and N-terminal sequencing, both on intact proteins and on their proteolytic fragments after trypsin digestion. Twenty-five peptide components of the proteome (including fiber) with greater than 0.25-0.5% contribution to the protein content of the virus were identified and characterized. Fiber was confirmed to be partially glycosylated (both the non-glycosylated and the monoglycosylated states were identified), and two proteins were isolated and identified as phosphorylation derivatives, namely protein V (non-phosphorylated and monophosphorylated) and protein IIIa (mono- and diphosphorylated). This new analytical tool proved to be very useful not only for refining our current knowledge of the polypeptide repertoire of purified infectious virions but also for monitoring and very rapidly identifying structural modifications resulting from changes in the manufacturing process. It was also used successfully for the characterization of various adenoviral constructs.