Objective: To evaluate the effect of water storage time on the cytotoxicity of soft liners. Methods: Sample discs of soft liners Dentusoft, Dentuflex, Trusoft, Ufi-Gel-P and denture base acrylic resin Lucitone-550 were prepared and divided into four groups: GN: No treatment, G24: Stored in water at 37°C for 24 h; G48: Stored in water at 37°C for 48 h, GHW: Immersed in water at 55°C for 10 min. To analyse the cytotoxic effect, three samples of each group were placed in tubes with Dubelcco's Modified Eagle Mediums and incubated at 37°C for 24 h. During this period, the toxic substances were leached to the culture medium. The cytotoxicity was analysed quantitatively by the incorporation of radioactivity 3H-thymidine checking the number of viable cells (synthesis of DNA). The data were statistically analysed using two-way anova and Tukey's honestly significant difference tests (α = 0.05). Results: Treatments did not reduce the cytotoxicity effect of the soft liners (p > 0.05). It was found that Ufi-Gel-P had a non-cytotoxic effect, Trusoft had a slightly cytotoxic effect, Dentuflex had a moderated cytotoxic effect, Dentusoft alternated between slightly and non-cytotoxic effect, and Lucitone-550 had non-cytotoxic effect when stored in water for 48 h. Conclusion: The effect of water storage and the heat treatment did not reduce the cytotoxicity of the soft liners. © 2011 The Gerodontology Society and John Wiley & Sons A/S.