Certain series of 2-thiohydantoin derivatives, carrying various substituents at position 5 such as 5-bromo-2-thienylmethylene, 5-(2-carboxyphenylthio)-2-thienylmethylene and 2-methylene-4H-thieno[2,3-b][1]benzothiopyran-4-one, were evaluated for their antitumor activity. Compound 5-(5-bromo-2-thienylmethylene)-3-morpholinomethyl-2-(2,3,4,6 -tetra-O-acetyl -beta-D-glucopyranosylthio)hydantoin proved to possess a broad spectrum antitumor activity against a wide range of different human cell lines of nine tumor subpanels causing both cytostatic and cytotoxic effects, resulting in full panel median growth inhibition (GI50) and total growth inhibition (TGI), with a median lethal concentration (LC50) at 15.1, 41.7 and 83.2 microM, respectively. On the other hand, compound 5-(5-bromo-2-thienylmethylene)-2-thiohydantoin and compound 5-(5-bromo-2-thienylmethylene)-3-phenyl-2- (2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-thio)hydantoin showed potential selectivity against leukemia cell lines. Further derivatization of these compounds, deduced from the obtained tentative structure-activity relationships, may lead to more potent agents.