The interaction of sanguinarine iminium form (structure I) and sanguinarine alkanolamine form (structure II) with calf thymus DNA has been studied in buffer of pH 5.2 and pH 10.5, respectively, where the physicochemical properties of DNA remain unchanged. The binding of sanguinarine iminium form to DNA is characterized by hypochromism and bathochromism in the absorption band, quenching of fluorescence intensity, increase in fluorescence polarization anisotropy, increase in positive and negative ellipticity of DNA, sign and magnitude of the thermodynamic parameters and increase in contour length of sonicated rodlike duplex DNA indicating that it binds to DNA by a mechanism of intercalation. In contrast, sanguinarine alkanolamine form does not show (i) any significant change in fluorescence polarization anisotropy, (ii) alteration of B form structure of DNA and (iii) increase in contour length of DNA indicating that it does not bind to DNA. But at a very high concentration of DNA, the alkanolamine form is influenced to form an iminium-DNA complex.