The effect of three dentin contacting materials on three-dimensional cultures of pulp-derived cells was evaluated in a dentin barrier test device. The test materials (Syntac Classic, Prompt L-Pop, Vitrebond) were applied on dentin disks of different thicknesses ranging from 100 to 500 microm. After 24 h of exposure with and without perfusion of the test chamber, cell survival was evaluated using the MTT assay and related to a nontoxic control material. Syntac Classic decreased cell activity significantly (p </= 0.0003), independently of the dentin thickness. For Prompt L-Pop and Vitrebond a significant influence of dentin thickness was found on the cell reaction. After exposure of the control material, photometric readings showed no dependency of the cellular reaction on dentin thickness (p > 0.05). It could be demonstrated that dentin acts as a barrier, decreasing the elicited cytotoxicity with increasing thickness. This effect is material related, showing little influence for the nontoxic or the glutaraldehyde containing material.