Purpose: To evaluate the cytotoxicity of three desensitizers, one nonrinse, and one etch-and-rinse adhesive system applied on dentin specimens of different thickness. Materials and Methods: The test materials (A: Admira Protect, B: Gluma Desensitizer, C: Seal&Protect, D: Clearfil Protect Bond, E: Optibond FL) and a positive control (35% H2O2) were applied on 1.0-, 1.5-, and 2.5-mm-thick bovine dentin specimens (each subgroup n = 5) in a dentin barrier test device. The experiments were performed with perfusion (2 ml/h) of the pulpal part of the chamber. The eluates were obtained before (baseline) and 15, 30, 45, 60, and 120 min after application of the adhesives and pipetted onto L-929 fibroblasts. Cytotoxicity of the materials was determined in relation to the baseline value using the MTT assay (succinic dehydrogenase activity). Statistical analysis was performed using ANOVA and Student's t-test. Results: With regard to 1.0-mm dentin specimens, application of Clearfil Protect Bond (D) decreased enzyme activity significantly, while test materials A to C and E were not cytotoxic. However, cytotoxicity of D was limited to up to 15 min and decreased thereafter. Application of the test materials A to E on 1.5- and 2.5-mm dentin samples exhibited no significant cytotoxic effects within 120 min. Generally, ANOVA found significant interactions between the test materials and dentin thickness. However, only for Admira Protect was a significant increase of enzyme activity with increasing dentin thickness observed. Conclusion: Desensitizing agents might exhibit cytotoxic potential when applied on dentin less than 1.0 mm thick. The tested desensitizers and the adhesive systems caused similar effects, in which cytotoxicty might be influenced by the duration of perfusion and dentin thickness. Keywords: desensitizer, cytotoxicity, dentin, perfusion, cell culture