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Abstract: Nonhuman adenoviruses including porcine adenovirus serotype 3 (PAd3) are emerging vectors for gene delivery. PAd3 efficiently transduces human and murine cells in culture, and circumvents preexisting humoral immunity in humans. The coxsackievirus–adenovirus receptor (CAR) serves as a primary receptor and αvβ3 or αvβ5 integrin as a secondary receptor for several human adenovirus (HAd) subtypes including HAd5. In this study, we deduced the role of CAR, αvβ3 or αvβ5 integrin in PAd3 internalization. Transduction experiments were conducted in human mammary epithelial (MCF-10A) cells using replication-defective PAd-GFP (PAd3 vector expressing green fluorescent protein [GFP]) and HAd-GFP (HAd5 vector expressing GFP). MCF-10A cells were treated with or without anti-human CAR, or anti-αvβ3 or anti-αvβ5 integrin antibodies prior to infection with HAd-GFP or PAd-GFP. Significant (P < 0.05) inhibition in transduction by HAd-GFP was observed in antibody-treated cells as compared to untreated cells, whereas transduction by PAd-GFP remained to similar levels irrespective of the treatment. To study the adenoviral fiber knob-mediated virus interference, MCF-10A cells were treated with or without the recombinant HAd5 or PAd3 knob followed by infection with HAd-GFP or PAd-GFP. Significant (P < 0.05) inhibition was observed only in transduction of the homologous vector. These results suggested that PAd3 internalization was CAR- as well as αvβ3 or αvβ5 integrin-independent and the primary receptor for HAd5 and PAd3 were distinct. CAR- and αvβ3 or αvβ5 integrin-independent entry of PAd3 vectors may have implications in targeting cell types that are not efficiently transduced by other adenoviral vectors. [Copyright &y& Elsevier]