Entry of human adenovirus into cells is a two-step process, mediated in the first step by a specific interaction between the trimeric fiber protein and a specific receptor on the surface of susceptible cells. Because of the interest in human adenovirus as a vector for gene therapy, we have mapped domains in the fiber protein that are important for proper assembly of this trimeric structure and for proper addition of O-linked N-acetylglucosamine (0-GlcNAc). Mutants of adenovirus type 2 fiber in this study were expressed in human cells by use of a recombinant vaccinia virus expression system that yielded protein indistinguishable from the fiber produced during adenovirus infection. The N-terminal half of the protein did not appear to influence fiber trimer formation, since deletions up to 260 amino acids (aa) from the N-terminal end as well as in-frame deletions within the shaft of the molecule still allowed trimerization; internal deletions in the shaft between aa 61 and 260 appeared to alter addition of 0-GlcNAc, as judged by loss of reactivity to a monoclonal antibody specific for this carbohydrate addition. Deletions from the C terminus of the molecule (as small as 2 aa) appeared to prevent trimer formation. Additions of amino acids to the C-terminal end of the fiber showed variable results: a 6-aa addition allowed trimer formation, while a 27-aa addition did not. These trimer-defective mutants were also relatively less stable, as judged kV pulse-chase experiments. Taken together, our results indicate that trimerization of the fiber requires at least two domains, the entire head (aa 400 to 582), and at least the C-terminal-most 15 aa of the shaft.