The development of methods for efficient and specific delivery of therapeutic genes into target tissues is an important issue for further development of in vivo gene therapy. In the present study, the physical targeting technique, photochemical internalization (PCI), has been used together with adenovirus. The combination of PCI and adenoviral transduction has previously been shown to be favorable compared to adenovirus used alone, and the aim of this study was to verify the role of the adenoviral receptors and identify the uptake pathway used by adenoviral particles in photochemically treated cells. All examined cell lines showed augmented transduction efficiency after PCI-treatment, with a maximum of 13-fold increase in transgene expression compared to conventionally infected cells. Blocking of CAR induced a complete inhibition of PCI-enhanced transgene expression. However, photochemical treatment managed to enhance the transduction efficiency of the retargeted virus AdRGD-GFP showing also that the virus-CAR interaction is not vital for obtaining a photochemical effect on adenoviral transduction. Blocking the alpha(V)-integrins reduced the gene expression significantly in photochemically treated cells. Subjecting HeLa cells expressing negative mutant-dynamin to light treatment after infection gave no significant increase in gene transfer, while the gene transfer were enhanced seven-fold in cells with wild-type dynamin. Furthermore, chlorpromazine inhibited photochemical transduction in a dose-dependent manner, whereas Filipin III had no effect on the gene transfer. In summary, the data presented imply that adenoviral receptor binding is important and clathrin-mediated endocytosis is the predominant uptake mechanism for adenoviral particles in photochemically treated cells.