The electrochemical oxidation of an anticancer drug, gemcitabine hydrochloride (GMB) at glassy carbon electrode has been studied by voltammetric techniques. GMB shows one irreversible oxidation peak at 0.927 V in phosphate buffer of pH 7.4. The effect of scan rate, pH, temperature, different electrolytes and surfactants on electrochemical behavior of GMB was investigated. Differential pulse voltammetry (DPV) and spectroscopic techniques viz., UV-vis absorption, circular dichroism and fluorescence were employed to probe the interaction between GMB and calf thymus DNA. From electrochemical data, the binding constant and binding ratio between DNA and drug are calculated to be 2.22 x 10(6) M(-1) and 1:2, respectively. Based on electrochemical and spectroscopic results, we concluded that the mode of binding of GEM to DNA was through groove binding. A DPV method with reasonable accuracy and precision was developed for the assay of GMB in the concentration range of 5 x 10(-6)-7.5 x 10(-4) M. The detection limit was observed to be 1.06 x 10(-6) M. The developed method was successfully applied to the determination of GMB in pharmaceutical formulations. The proposed Study may serve as a reference for the interaction of GEM with DNA base pairs in the natural environment of living cells. (C) 2009 Elsevier B.V. All rights reserved.