The purpose of this study was to investigate the influence of different methods of skin preservation on the percutaneous permeation of two drugs with varied physicochemical properties (melatonin and nimesulide). Hairless rat skin was freshly excised and immediately stored at three different storage conditions: (a). 4 degrees C in minimal essential medium eagle (MEM), (b). frozen at -22 degrees C and (c) frozen at -22 degrees C in 10% glycerol (as a cryoprotective agent). The permeation of melatonin and nimesulide from saturated solutions was studied using the skin stored at various cold storage conditions for 6 months. At 4 degrees C, the flux of melatonin was similar to fresh skin for up to 7 days (P>0.05) and increased 2.4-fold at 14 days (P<0.001). The flux of melatonin was similar to fresh skin for 14 days with skin at -22 degrees C (P>0.05) and then steadily increased from 30 days onwards and at 180 days, the flux was 5-fold greater than fresh skin (P<0.001). Freezing of skin at -22 degrees C with 10% glycerol showed similar flux values for melatonin up to 60 days and at 180 days the flux increased 2.2-fold as compared to fresh skin (P<0.001). In the case of nimesulide, the skin stored for 2 days at 4 degrees C showed similar flux as compared to fresh skin (P>0.05) and increased steadily from 4 days onwards and at 14 days the flux was 3.5-fold higher than fresh skin (P<0.001). The skin frozen at -22 degrees C with or without 10% glycerol showed no difference in flux up to 4 days which however increased from 7 days onwards. At 180 days, the skin at -22 degrees C showed 2.5-fold increase in the flux (P<0.001) whereas the skin frozen with 10% glycerol showed 2-fold increase in the flux (P<0.001) as compared to fresh skin. The results of the present study demonstrate that the permeability of the drugs across the stored skin was dependent on the storage condition, the length of storage and the physicochemical properties of the drug under study. Overall, the freezing of skin at -22 degrees C with 10% glycerol was found to be very helpful for the long-term storage of skin for percutaneous permeation studies.