In order to analyze the role of alpha(v)beta(3) and alpha(v)beta(5) integrins in gene transfer by adenovirus-based vectors, an RGD4C motif was inserted into the HI-loop of wild type or shortened fiber protein of human adenovirus of serotype 5, thereby creating Ad5RGD4C and Ad5Delta639RGD4C vectors, respectively. Infection by the latter is independent of the Coxsackie B adenovirus receptor. Internalization and transduction of these vectors and were investigated in several stably transfected cell clones derived from a human laryngeal carcinoma cell line (HEp2) expressing different ratios of alpha(v)beta(5) and alpha(v)beta(3) integrins. We show that alpha(v)beta(3) is more successful than alpha(v)beta(5) in: (i) mediating adenovirus internalization and transduction when the RGD motif is present only in the penton base and (ii) mediating internalization and transduction by RGD4C-fiber modified adenoviruses. The highest amount of internalized virus was found in the cell clone in which alpha(v)beta(3) integrin predominated over alpha(v)beta(5) integrin (as judged by the % of cells expressing alpha(v)beta(3) and alpha(v)beta(5) integrins). However the level of transgene expression in this cell line was even lower than that in parental HEp2 cells which do not express alpha(v)beta(3) integrin. This discrepancy between internalization and transgene expression (transduction) is likely due to the crucial role of alpha(v)beta(5) in membrane permeabilization, indicating that alpha(v)beta(5) integrin is a limiting factor for Ad5-mediated gene transfer. We conclude that alpha(v)beta(3) integrin is an efficient adenovirus internalization receptor, but cannot functionally replace alpha(v)beta(5) in endosomal release.