The cytotoxicity of three root-end filling materials (amalgam, IRM, and Super-EBA) was evaluated in cultures of human periodontal ligament cells and human osteoblast-like cells. Ten-millimeter-long plastic test tubes were filled with 3 mm of freshly mixed root-end filling materials at one end (1.5 mm diameter). The opposite end was sealed and attached by heat to a 35-mm cell culture dish. Human periodontal ligament cells and human osteoblast-like cells were seeded in the dishes. The size of cell-free zones around the root-end filling materials and the total cell number per dish were calculated after 3 and 7 days. Empty test tubes used as controls did not influence the growth and distribution of the cultured cells. Cell density increased in all groups in the test period. Amalgam had a larger cell-free zone, compared with IRM and Super-EBA and showed a reduction in total cell number per dish for both tested cell types. IRM and Super-EBA also had a cell-free inhibition zone for both cell types, but no significant reduction in total cell number per dish. This study showed that amalgam had a higher cell toxicity to human periodontal ligament cells and human osteoblast-like cells than IRM and Super-EBA.