A buccal physiological in vitro testing system for the evaluation of the permeability, the transport route and toxic effects of nanoparticles was developed. Carboxyl polystyrene (CP, 20 nm, 200 nm) and amine modified polystyrene (AP, 200 nm) particles were used as reference particles and characterized in biological media. The permeability through excised porcine buccal mucosa was investigated with Franz diffusion cells. To evaluate the transport route, particle uptake into oral H376 cells was recorded and the cell damage was measured. All particles immediately formed aggregates once dispersed in saliva. 20 nm CP particles permeated the mucus layer and penetrated into the stratum superficiale of the top third region of the epithelium by the transcellular route. The positively-charged 200 nm AP particles permeated the mucus-layer and penetrated into deeper regions of the tissue. By decreasing the temperature to 4°C, particle uptake was inhibited for 20 nm CP and 200 nm AP particles. 200 nm CP particles interacted with the mucus, formed agglomerates and did not penetrate into the epithelium. It can be concluded that the presented system serves as a valuable tool to evaluate the behavior of nanoparticles in the buccal mucosa.