3-substituted-2-thiohydantoin derivatives were synthesized and their structures elucidated by IR, 1H-NMR spectroscopy and elemental analysis. The cytotoxicity of the 2-thiohydantoin derivatives to rat embryo fibroblasts (F2408) in vitro was determined, and the effects of these compounds on intracellular free Ca2+, [Ca2+]i, were measured by spectrofluorophotometry. Cytotoxicity was determined by metabolic reduction of a tetrazolium salt to a formazan dye (MTT assay). Compounds 4 and 7 showed cytotoxic activity, with IC50values in the range of 1–1.2 μM. Introduction of either chlorophenyl, metoxyphenyl, nitrophenyl or benzyl groups at C-3 resulted in concentration-dependent cytotoxic effects. Compounds 1–6 at 1 μM or more significantly increased [Ca2+]iin a dose-dependent manner in the cultured fibroblasts. This action may have been mediated through intracellular calcium stores.