Photochemical internalization (PCI) enhances adenovirus (Ad) transgene expression in a variety of cell lines in vitro. However, measurements of the photochemical effect on transduction in multicellular environments are lacking. In this study, spheroids of DU 145 prostate cancer cells were used as a model to evaluate Ad serotype 5 (Ad5) transduction in a multicellular environment in response to PCI treatment. Furthermore, the Ad5 was coated with poly(2-methyl-acrylic acid 2-[(2-(dimethylamino)-ethyl)-methyl-amino]-ethyl ester) (pDAMA) to evaluate whether physicochemical properties such as charge and size of viral vectors affect transduction of photochemically treated spheroids. Spheroids incubated with photosensitizer TPPS(2a) (1 microg ml(-1)) and infected with adenovirus contained 3-fold higher percentage of reporter gene expressing cells after exposure to blue light (0.42 J cm(-2)) compared to no light, as analysed by flow cytometry of dissociated spheroids two days after treatment. The cells within the infected spheroids were further divided into three sections corresponding to the interior, intermediate and peripheral layers of the spheroids. This was performed by staining the spheroids with a diffusion-limited dye prior to dissociation. Transduction of cells within photochemically treated and untreated spheroids was heterogeneous, with a radial reduction of transgene expression towards the inner section of the spheroid. The coating of Ad with pDAMA induced up to 2-fold decrease in transduction of cells in the interior section of spheroids compared to uncomplexed Ad, while transduction of the peripheral section remained unchanged. The decrease in transduction could be related to reduced diffusion due to the size of the Ad-pDAMA complexes.