Purpose: Adenovirus serotype 5 (Ad5) has been used for gene therapy with limited success due to insufficient infectivity in cells with low expression of the primary receptor, the coxsackie and adenovirus receptor (CAR). Evidence that adenovirus serotype receptors other than CAR may be of use was presented in previous studies that showed that the Ad3 receptor is expressed at high levels in ovarian cancer cells. We hypothesized that combined use of unique chimeric fibers in the context of novel mosaic adenovirus vectors would enhance infectivity via non-CAR pathways in ovarian cancer cells. Experimental Design: We constructed and characterized Ad5 vectors that use Ad3 knob and reovirus fibers to generate a mosaic fiber virion. Serotype 3 Dearing reovirus uses a fiber-like σ1 protein to infect cells expressing sialic acid and junction adhesion molecule 1. We therefore constructed a mosaic fiber Ad5 vector, designated Ad5/3-σ1, encoding two fibers: a σ1 chimeric fiber and the chimeric Ad5/3 fiber composed of an Ad3 knob. Results: Functionally, Ad5/3-σ1 used sialic acid, junction adhesion molecule 1, and Ad3 receptor for cell transduction and achieved maximum infectivity enhancement in ovarian cancer cells with low CAR expression. Furthermore, Ad5/3-σ1 achieved infectivity enhancement in primary tissue slices of human ovarian tumor. Conclusions: We have developed a new type of Ad5 vector with the novel tropism, possessing fibers from Ad3 and reovirus, which exhibits enhanced infectivity via CAR-independent pathway(s). In addition, the flexible genetic platform of vector allows different combination of fiber variants that can be incorporated within the same particle. © 2007 American Association for Cancer Research.