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Five root\u2010end filling materials were compared regarding the cytotoxicity, apoptosis, and mitochondrial dehydrogenase (MDH) activities of human periodontal ligament (PDL) fibroblasts, with the use of a novel transwell culture system. Exposure to IRM (a ZnO eugenol\u2010based intermediate restorative material), a 2\u2010ethoxybenzoic acid cement (Super EBA), and amalgam for 3 days inhibited the MDH activity of PDL fibroblasts as indicated by decrease in 3\u2010(4,5\u2010dimethylthiazol\u20102\u2010yl)\u20102,5\u2010diphenyl\u2010tetrazolium bromide (MTT) reduction by 97%, 95%, and 51%, respectively. Evident suppression of MTT reduction by amalgam and glass ionomer cement (GIC) was noted after 5 days of exposure, with 73% and 46% of inhibition, respectively. Mineral trioxide aggregates (MTA) showed little effect on MDH activity. IRM and Super EBA were cytotoxic to PDL fibroblasts as indicated by a trypan blue dye exclusion technique. GIC and amalgam showed mild cytotoxicity. IRM, GIC, and amalgam further induced apoptosis of PDL cells, as revealed by the presence of sub\u2010G0\/G1 DNA content in flow cytometric histogram. Twenty\u2010four\u2010hour exposure to IRM and Super EBA elevated the MDH activities to 156% and 117%, correspondingly, of that of control. Eugenol, a phenolic ingredient in Super EBA and IRM, also increases MDH activity of PDL fibroblasts by 45% and 51%, at concentrations of 0.5 and 1 m<jats:italic>M<\/jats:italic>. However, at concentrations higher than 0.5 m<jats:italic>M<\/jats:italic>, eugenol decreased the number of viable PDL fibroblasts. These results suggest that MTA is a biocompatible root\u2010end filling material, followed by self\u2010curing Fuji II GIC and amalgam. IRM and Super EBA ingredients induced marked cytotoxicity and transiently stimulate MDH activities, which is possibly due to their content of eugenol and induction of cellular adaptive response. \u00a9 2004 Wiley Periodicals, Inc. 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