A study of the relationship between the chemical structure of cytokinins and their cytotoxic effects against a panel of human cancer cell lines with diverse histopathological origins is presented. Test compounds included almost all known natural cytokinins (N = 42) and two groups of novel synthetic analogues: derivatives of N6-benzyladenosine with diverse substitutions on the benzyl ring (N = 48) and their analogues where the ribose moiety is replaced by a tetrahydropyran-2-yl or tetrahydrofuran-2-yl group (N = 34). Strong cytotoxic activity was limited to certain cytokinin ribosides and their corresponding ribotides. The anticancer activity of aromatic cytokinin ribosides can be improved by fluorination or ortho- hydroxylation of the benzyl ring. The potent anticancer activity of the natural cytokinin ortho-topolin riboside (median GI50 = 0.65 ?M) was confirmed using NCI60. Its activity pattern was distinctly different from those of standard anticancer drugs, suggesting that it has a unique mechanism of action. In comparison with standard drugs, ortho-topolin riboside showed exceptional cytotoxic activity against NCI60 cell lines with a mutated p53 tumour suppressor gene. Ortho-topolin riboside also exhibited significant anticancer activity against several tumour models in in vivo hollow fibre assays.A study of the relationship between the chemical structure of cytokinins and their cytotoxic effects against a panel of human cancer cell lines with diverse histopathological origins is presented. Test compounds included almost all known natural cytokinins (N = 42) and two groups of novel synthetic analogues: derivatives of N6-benzyladenosine with diverse substitutions on the benzyl ring (N = 48) and their analogues where the ribose moiety is replaced by a tetrahydropyran-2-yl or tetrahydrofuran-2-yl group (N = 34). Strong cytotoxic activity was limited to certain cytokinin ribosides and their corresponding ribotides. The anticancer activity of aromatic cytokinin ribosides can be improved by fluorination or ortho- hydroxylation of the benzyl ring. The potent anticancer activity of the natural cytokinin ortho-topolin riboside (median GI50 = 0.65 ?M) was confirmed using NCI60. Its activity pattern was distinctly different from those of standard anticancer drugs, suggesting that it has a unique mechanism of action. In comparison with standard drugs, ortho-topolin riboside showed exceptional cytotoxic activity against NCI60 cell lines with a mutated p53 tumour suppressor gene. Ortho-topolin riboside also exhibited significant anticancer activity against several tumour models in in vivo hollow fibre assays.