The mucus layer is believed to play a part in drug permeation across the oral mucosa. Human freeze-dried saliva (HFDS) and porcine gastric mucin (PGM) was evaluated as model for mucus layer per se or in conjunction with in vitro and ex vivo buccal permeability models. Four small molecules (nicotine, mannitol, propranolol, caffeine) showed decreased permeability across mucin dispersions, compared to controls, and a greater effect was seen with HFDS than with PGM. Permeability of propranolol and caffeine across filter-grown TR146 cells was decreased by the presence of mucin, whereas no effect was found on nicotine and mannitol. Incubation of porcine buccal mucosa with mucin dispersions for 24 h compromised the integrity of the tissue, whereas 30 min incubation did not affect tissue integrity. Tissue incubation with mucin dispersions did not decrease nicotine permeability. For the studied model drugs, it is concluded that mucin dispersions constitute a minor barrier for drug diffusion compared to the epithelium.