Peroral nanoparticle-mediated drug absorption was studied using a laser scanning confocal microscope. Additional diffusion studies in side-by-side diffusion cells with radiolabelled polybutylcyanoacrylate (PBCA) nanoparticles were carried out to confirm the results of this study. Fluorescence-labelled PBCA nanoparticles were incubated in vitro in the lumen of freshly excised intestine. Computer-aided optical sectioning of thick samples with dramatically improved resolution and the possibility of rejecting out-of-focus noise enabled tracking of the fluorescence-labelled PBCA nanoparticles in the intestinal tissue after incubation of the particles in freshly excised porcine small intestine. The results of this study suggest that the nanoparticles are absorbed by the surface of the gut wall, creating a high concentration gradient, thereby enhancing the absorption of drugs that may be loaded to the nanoparticles. A significant amount of particles was found in hot (very fluorescent) spots that were assumed to be Peyer's patches. No particles, however, traversed the entire gut wall over a period of 2 to 4 h. These results were confirmed by the diffusion study. No radioactivity permeated through Peyer's-patch-free intestine within 4 h, whereas the amount of radioactivity that was transported through intestine with Peyer's patches during this time was 1.1% of the total amount in the donor chamber.