Hoechst 33258 belongs to bisbenzimidazole class of molecules having anticancer properties for their ability to inhibit topoisomerase and many other cellular processes. The aim of the present study is to understand the nature of Hoechst 33258-bovine serum albumin (BSA) binding interactions by using absorption, fluorescence and circular dichrorism (CD) measurements under simulative physiological conditions. The absorption spectra of BSA indicated the binding of Hoechst 33258 with BSA. The analysis of fluorescence data indicated the presence of both dynamic and static quenching mechanism in the binding. The associative binding constant and number of binding sites were found to be K=2.08=107M-1 and n=1.36 respectively. Biexponential fluorescence lifetime distribution of Hoechst 33258 in the presence of BSA has altered viz. t1 was increased significantly from 0.3 ns (60%) to 1.2 ns (13%) whereas a marginal increase in t2 from 3.6 ns (40%) to 4.0 ns (87%). Fluorescence anisotropy value of Hoechst 33258 has increased from 0.14 to 0.34 upon the addition of BSA. Thermodynamic parameters were also calculated using Van't Hoff plot by conducting fluorescence titration at four different temperatures, ΔH=+102.785 kJ mol-1, ΔS=+490.18 kJ mol-1, ΔG=+491.708 kJ mol-1. The CD spectrum of BSA revealed that the binding of Hoechst 33258 to BSA causes loss in the secondary structure but increases the thermal stability of the protein. The results indicated that hydrophobic interactions were the predominant intermolecular forces in stabilizing BSA-Hoechst 33258 complex. The possible implications of these results will be on designing better therapeutic minor groove binding drug molecules. © 2009 Pharmaceutical Society of Japan.