A new series of 3,6-disubstituted triazolo[3,4-b]thiadiazole derivatives have been synthesized by simple, high yielding routes. The key step in the construction of the triazolo[3,4-d]thiadiazole nucleus involves the reaction of 4-amino-5-substituted [1,2,4]triazole-3-thiol with carbon disulphide, 4-amino benzoic acid, (2-amino[1,3]thiazole-4-one-5-yl) acetic acid, and (1H-pyrazolo[3,4-d]pyrimidine-2,4-dithione-5-yl) acetonitrile. The newly synthesized compounds were evaluated for their cytotoxic activity against a panel of 60 human cancer cell lines by the National Cancer Institute (NCI) and some of them demonstrated inhibitory effects on the growth of a wide range of cancer cell lines generally at 10(-5)M level and in some cases at 10(-7)M concentrations. In this assay, the anti-tumor activity of the newly synthesized compounds could not be interpreted in terms of tyrosine kinase inactivation but more likely as a relatively broad specificity for the ATP-binding domain of other kinases. The pharmacological mechanism of action for these intriguing compounds has not, as yet, been successful.