Adenovirus serotype 5 (Ad5) displays unparalleled gene transfer efficacy to cells with high coxsackie-adenovirus receptor (CAR) expression. Unfortunately, cells isolated from clinical human cancers, both ovarian and other types, express highly variable and often low levels of CAR. Fortunately, native Ad5 tropism can be modified to circumvent CAR deficiency and to enhance infectivity. Ad5/3luc1 incorporates the serotype 3 fiber knob and binds to a receptor distinct from CAR, while the fiber of Ad5lucRGD is modified with an RGD-4C motif, allowing CAR-independent binding to integrins. We studied the liver tropism and blood clearance of these viruses after intravenous (i.v.) injection, and biodistribution after intraperitoneal (i.p.) injection to tumor-bearing mice. To estimate efficacy, we assessed gene transfer to purified human primary ovarian cancer cells, and in a mouse model of ovarian cancer. Ad5/3luc1 achieved improved gene transfer over Ad5lucRGD, and both infectivity-enhanced viruses were superior to the isogenic control with an unmodified Ad5 capsid. In the presence of malignant ascites, gene transfer was improved with both Ad5/3luc1 and Ad5lucRGD. Thus, retargeting to the Ad3 receptor enhances gene transfer to clinically relevant ovarian cancer substrates, while the mouse toxicity and biodistribution profile of both fiber-modified Ad vectors is comparable to Ad5.