Attachment of human adenoviruses (Ads) to host cells is mediated by the interaction of the fiber protein of the capsid with specific cell-surface molecules. For one of the species B adenoviruses, Ad3, the mechanism of binding to cells remains to be defined. Several previous reports have proposed CD46, CD80 or CD86 as possible Ad3 fiber attachment molecules. In this study, CD80 and CD86 were not found to mediate Ad3 fiber binding or Ad3-EGFP transduction of cells. Low levels of Ad3-EGFP transduction of a CHO cell line expressing relatively high levels of CD46 were detected which might suggest a role for CD46 in facilitating Ad3: cell interactions, in the absence of other attachment molecules. Anti-CD46 antibodies and siRNAs had almost no effect on Ad3 fiber binding or Ad3-EGFP transduction of HeLa cells. However, treatment of A549 cells with CD46 siRNA resulted in some decrease of transduction with Ad3-EGFP.