The estrogenic activity of phthalate esters for dental use as plasticizers was concerned. To study the feasibility of candidate chemicals for phthalate tissue conditioners, three candidate plasticizers, di-n-butyl sebacate, acetyl tributyl citrate and tri-n-butyl phosphate together with conventional plasticizers were examined for estrogenic activity and cytotoxicity using three estrogenicity assays, human gingival fibroblasts and living skin equivalent. None of the three candidate plasticizers showed any estrogenic activity at the concentrations tested by the three assays. Di-n-butyl sebacate, acetyl tributyl citrate and their prototype tissue conditioners were weakly to negligibly cytotoxic, while tri-n-butyl phosphate and its prototype tissue conditioner yielded the strongest cytotoxicity among the six plasticizers tested. In particular, the cytotoxicities of di-n-butyl sebacate and its prototype tissue conditioners were significantly weaker than the others and the conventional plasticizers. The results suggested that di-n-butyl sebacate and acetyl tributyl citrate could be candidate chemicals for phthalate ester-free tissue conditioners.