In this study the interaction of daunomycin with HMGB1 nonhistone chromatin protein in the chromatin context using hydroxyapatite (HAP) column chromatography and free in solution was investigated employing fluorescence, circular dichroism spectroscopy and thermal denaturation techniques. The results demonstrate that HMGB1 fraction eluted from HAP column contained the most amount of daunomycin. Upon addition of daunomycin to HMGB1 solution, fluorescence emission intensity was dependent on the drug concentration used whereas the ellipticity in CD spectra was decreased at both 205 and 220 nm extremes implying that quenching of the drug with the HMGB1 chromospheres alters secondary structure of the protein. Although daunomycin slightly increased the melting point of HMGB1, but exhibited a significant hyperchromicity at low concentrations and hypochromicity at higher concentrations of daunomycin. The results suggest that daunomycin binds to HMGB1 protein which may influence its interaction with DNA in nucleosomes and other cellular processes.