Objectives: To compare the cytotoxicity of the polymerized bonding agents Scotchbond 1 (3M ESPE), Prime & Bond NT (Dentsply DeTrey), Xeno III (Dentsply DeTrey), and Clearfil Protect Bond (primer and bond parts; Kuraray) on mouse fibroblast cells. Xeno III was also tested through thin dentin disks. Method and Materials: Near-confluent 3T3 cells were exposed to Dulbecco modified eagle's medium extractions from the above-mentioned agents, and the cell viability (survival rate) was measured using the standard MTT assay and related to the nonexposed controls. Results: All bonding agents were found to be cytotoxic toward the 3T3 cells. Scotchbond 1 (59% survival rate) and Prime & Bond NT (62% survival rate) were not statistically different (Kruskal-Wallis test, P > .05). However, the survival rates of Xeno III (25% through membrane and dentin disks) and Clearfil Protect Bond (35%) were significantly lower than those of the other 2 bonding agents, with Xeno III significantly the most toxic (Kruskal-Wallis test, P .05). Of the 2 parts from Clearfil Protect Bond, the bond part was the least toxic (91% survival rate), but the primer part was very toxic (30% survival rate). Conclusion: In an in vitro culture, all 4 dentin bonding agents were cytotoxic. Xeno III was the most toxic, even through dentin disks. It seems that the cytotoxicities depend on the compositions of the materials tested. The most toxic part of Clearfil Protect Bond was the primer, which contains the antibacterial pyridinium molecule. Keywords: bonding agents, cytotoxicity, mouse fibroblast