Adenovirus serotype 5 (Ad5) has been used for gene therapy with limited success because of insufficient infectivity in cells with low expression of the primary receptor, the coxsackie and adenovirus receptor (CAR). To enhance infectivity in tissues with low CAR expression, tropism expansion is required via non-CAR pathways. Serotype 3 Dearing reovirus utilizes a fiber-like σ1 protein to infect cells expressing sialic acid and junction adhesion molecule 1 (JAM1). We hypothesized that replacement of the Ad5 fiber with σ1 would result in an Ad5 vector with CAR-independent tropism. We therefore constructed a fiber mosaic Ad5 vector, designated as Ad5-σ1, encoding two fibers: the σ1 and the wild-type Ad5 fiber. Functionally, Ad5-σ1 utilized CAR, sialic acid, and JAM1 for cell transduction and achieved maximum infectivity enhancement in cells with or without CAR. Thus, we have developed a new type of Ad5 vector with expanded tropism, possessing fibers from Ad5 and reovirus, that exhibits enhanced infectivity via CAR-independent pathway(s). © 2005 Elsevier Inc. All rights reserved.