The interaction between DNA and several newly synthesized derivatives of the natural anticancer compound luotonin A has been studied. The results from our work reveal an effective and selective alkaloid/double-stranded DNA (ds-DNA) interaction. In the presence of increasing amounts of ds-DNA, a noticeable fluorescence quenching of the luotonin A derivatives under study was observed. However, this effect did not take place when single-stranded DNA (ss-DNA) was employed. The association constant alkaloids/ds-DNA was calculated by quantitation of such a quenching effect. The influence of other quenchers, namely Co(2+) and Br(-) on the native fluorescence of luotonin A and derivatives was also studied, and a remarkable quenching effect was observed for both ions. We have also investigated how by binding DNA the alkaloids could get protected from the external Co(2+) and Br(-) quenchers. The Stern-Volmer constants (K (SV)) for Co(2+) and Br(-) quenching effect on the studied alkaloids were considerably reduced (10-50%) after incubation of the compounds in the presence of DNA with regard to the K (SV) values in absence of DNA. An increase in the fluorescence anisotropy values of luotonins was also produced only in the presence of ds-DNA but not in the case of ss-DNA. To better characterize the nature of that interaction, viscosimetry assays and ethidium bromide displacement studies were conducted. With regard to DNA reference solutions, the viscosity of solutions containing DNA and luotonin A derivatives was reduced or not significantly increased. It was also observed that the studied compounds were unable to displace the intercalating agent ethidium bromide. All of these results, together with the obtained association constants values (K (ass) = 2.2 × 10(2) - 1.3 × 10(3)), support that neither covalent nor intercalating interactions luotonin A derivatives/ds-DNA are produced, leading to the conclusion that these alkaloids bind ds-DNA through the minor groove. The specific changes in the fluorescence behavior of luotonin A and derivatives distinguishing between ss-DNA and ds-DNA binding, lead us to propose these compounds as attractive turn-off probes to detect DNA hybridization.