Aim: To investigate the cytotoxicity and estrogenicity of Vivera® retainers by assessing their biological behavioral effects as-received from the manufacturer and after retrieved from patients. Materials and methods: In this, in vitro investigation six sets (maxillary and mandibular) of Vivera® retainers, three as received and three retrieved after four weeks of use by patients of an orthodontic postgraduate clinic, were immersed in the normal saline solution for 14 days following different modes of sterilization. The estrogenicity assays involved two cell lines, namely the estrogen-sensitive MCF-7 and the estrogeninsensitive MDA-MB-231. Following a 6 day incubation with the solutions to be tested, at concentrations varying from 5% to 20% v/v in medium supplemented with 2% fetal calf serum devoid of endogenous estrogens, estrogenicity was assessed by cell counting; β-Estradiol was used as positive control. The statistical analysis of data was performed with two-way analysis of variance (ANOVA) with appliance and concentration as predictors. Differences were further investigated with the Tukey multiple comparison tests at the 0.05 level of significance. Results: No significant MCF-7 proliferation was induced by the three samples compared either to the eluents from as-received retainers or to the negative control. As expected, β-estradiol induced a potent stimulation of MCF-7 cell proliferation, while no effect was observed on MDA-MB-231 cells. Conclusion: Under the conditions of this experiment eluents of as-received and retrieved Vivera® retainers did not seem to exhibit xenoestrogenic activity. Clinical significance: Vivera® retainers can be used as parttime removable oral appliances following the manufacturer's instructions.