INTRODUCTION: Along with introduction of dentin bonding agents (DBA), their clinical use as lining materials is increasing rapidly. Since remaining dentinal thickness (RDT) has always been a concern for cytopathic effect of restorative materials, its effect on reduction of cytotoxicity of these materials especially DBAs is critical. The purpose of this study was to evaluate and compare the cytotoxicity of three dentin bonding systems, belonged to the 4th, 5th and 6th generation of DBAs on L929 cell line. MATERIALS AND METHODS: Thirty human premolar teeth were included. Class I cavity preparations were prepared on occlusal surfaces. After crown separation, a flat dentinal surface was provided and RDT (remaining dentinal thickness) was adjusted at 0.5 and 1.5 mm. Then, cavities were treated in three groups with experimental DBAs: Group 1: Scotchbond multipurpose, Group 2: Excite, Group 3: AdheSE. Blue inlay wax sealed the cavities. Crowns were immersed in culture medium for 24 hours and the cytotoxicity of the resultant toxic medium was measured quantitatively with MTT assay in 4 serial dilutions. Data were analyzed with ANOVA and Tukey’s test at 95% significance level. RESULTS: MTT assay determined that only in neat dilution of 0.5 mm RDT, cell changes were significantly different from control. Besides, no significant differences were found between the three experimental DBAs regarding cytotoxic effect on L929 cell line. CONCLUSION: Considering the limitations of an in vitro study, if the RDT is less than 0.5 mm in vivo, regardless of the type of DBA, destructive cellular changes in pulp tissue can be expected.