Entry of the adenovirus (Ad) capsids during the early stages of infection is a multistep process that includes initial attachment of the virus capsid to the cell surface followed by internalization of the virus into early endosomes. The Ad fiber protein, a complex of three apparently identical subunits, mediates the initial attachment step. In this chapter, methods for the purification and characterization of the Ad fiber protein are presented. Chromatographic methods for the isolation of the protein from infected cells can yield substantial quantities of protein for biochemical analysis. Protocols for characterization of the protein by Western blot and by indirect immunofluorescence of infected cells are also presented. The specificity of different monoclonal and polyclonal antibodies that recognize Ad fiber is also discussed. Ad fiber from a number of serotypes also contains a posttranslational modification, O-linked N-acetyl-glucosamine; methods for detection and characterization of this modification are also provided. With these tools and protocols, one can address important questions about this protein, which helps direct the tissue tropism of Ad.