Ex vivo drug permeation studies are useful for early screening of drug candidates for buccal delivery. However, it is not always feasible to obtain fresh tissue for each experiment. Therefore, a method for storing excised tissue for later use is needed. The purpose of this study was to determine if permeability barriers for small molecules (nicotine and diazepam) were maintained after freezing porcine buccal mucosa with cryoprotectants to -80°C. Combinations of dimethyl sulfoxide, bovine serum albumin, glycerol and sucrose were used as cryoprotectants. The permeability of nicotine and diazepam across fresh or frozen/thawed tissue was determined using modified Ussing chambers. Haematoxylin-eosin stained tissue sections for histology were prepared. The permeability of nicotine across tissue frozen without cryoprotectants was significantly higher compared to tissue frozen with cryoprotectants or fresh tissue. Freezing with or without cryoprotectants did not significantly affect the flux of diazepam compared to fresh tissue. Only minor histological changes were seen in frozen/thawed porcine buccal mucosa compared to fresh tissue. In conclusion, permeability barriers for nicotine and diazepam were preserved after freezing with any of the combinations of cryoprotectants; however, the barrier may be damaged when freezing without cryoprotectants.