The purpose of this study was to compare the influence of three kinds of storage methods on surface roughness of tissue conditioners. Four commercial tissue conditioners (GC Soft Liner, Softone, Fictioner, and Hydro-Cast) were used in this study. Five samples of each material were stored in distilled water, air, and a denture cleanser (Polident). Mean surface roughness (Ra) values of dental stone casts made from the tissue conditioners were measured after 0, 1, 3, 7, and 14 days of immersion using a profilometer. Significant differences in the Ra values of the specimens were found among the three storage methods. The values of Ra significantly increased with increase in immersion time for each storage method, except for the materials stored in air. It was found that the materials stored in air showed the most stable and lowest values of Ra. Results obtained suggested that a tissue conditioner exhibited smooth and minimal change in surface roughness with time when stored in air than in distilled water and denture cleanser.