Adenoviruses replicate primarily in the host cell nucleus and it is well established that adenovirus infection affects the structure and function of host cell nucleoli in addition to coding for a number of nucleolar targeted viral proteins. Here we have used unbiased proteomic methods, including high throughput mass spectrometry coupled with Stable Isotope Labeling of Amino acids in Cell culture (SILAC) and traditional two dimensional (2-D) gel electrophoresis to identify quantitative changes in the protein composition of the nucleolus during adenovirus infection. 2-D gel analysis revealed changes in six proteins. By contrast, SILAC based approaches identified 351 proteins with 24 proteins showing at least a two-fold change after infection. Of those, four were previously reported to have aberrant localisation and/or functional relevance during adenovirus infection. In total, 15 proteins identified as changing in amount by proteomic methods were examined in infected cells using confocal microscopy. Eleven of these proteins showed altered patterns of localisation in adenovirus-infected cells. Comparing our data with the effects of actinomycin D on the nucleolar proteome revealed that adenovirus infection apparently specifically targets a relatively small subset of nucleolar antigens at the time point examined.