Common human adenovirus (Ad) vectors are derived from serotype 2 or 5, which use the coxsackie-adenovirus receptor (CAR) as their primary cell receptor. We investigated the receptor usage of mouse adenovirus type 1 (MAV-1), which in vivo is characterized by a pronounced endothelial cell tropism. Alignment of the fiber knob sequences of MAV-1 and those of CAR-using adenoviruses, revealed that amino acid residues, critical for interaction with CAR, are not conserved in the MAV-1 fiber knob. Attachment of MAV-1 to Chinese hamster ovary (CHO) cells was not increased by stable transfection with mouse CAR, whereas the binding efficiency of Ad2 was 20-fold higher in the mouse CAR-transfectant compared to the wild type cells. Also, purified fiber knob of Ad5, which is interchangeable with the Ad2 fiber knob, did not compete with MAV-1 for receptor binding, indicating that MAV-1 binds to a receptor different from CAR. These results support further exploration of an MAV-1-derived vector as a potential vehicle for gene delivery to cell types which are not efficiently transduced by human adenovirus vectors.