Adenovirus (Ad) fiber proteins are responsible for the initial attachment of the virion to the cell membrane. Most Ad vectors currently in use are based on the Ad type 5 (Ad5), which belong to subgroup C, and use the coxsackievirus and adenovirus receptors (CAR) as the initial receptor. Ad35, which belongs to subgroup B, recognizes unknown receptor(s) other than CAR. In this study, the feasibility of the Ad vector containing Ad5/35 chimeric fiber protein was examined in a wide variety of cell types, such as CAR-positive or -negative human tumor cells, rodent cells, and blood cells (a total of 20 cell types), and in mice in vivo. Transduction data suggested that the Ad vectors containing the Ad5/35 chimeric fiber protein exhibited altered and expanded tropism when compared with the Ad5-based vector. The chimeric vector also allows the packaging of larger foreign DNAs than the conventional Ad5-based vector, which can package approximately 8.1-8.2 kb of foreign DNA. The chimeric vector containing approximately 8.8 kb of foreign DNA was generated without affecting the viral growth rate and titer. These results suggested that inclusion of the Ad35 fiber protein into the Ad5-based vector could lead to an improved efficiency in gene therapy and in gene transfer experiments, especially for the cells lacking in sufficient CAR expression.