Abstract Three large fragments (A, B and C) of human serum albumin (HSA) were produced by cyanogen bromide digestion of HSA in order to investigate the specific binding sites. The fragments were isolated by use of gel filtration, followed by high performance ion exchange chromatography. The isolated fragments were examined by use of UV/Vis, steady-state fluorescence, and circular dichroism spectroscopy. The study was extended to examine the interactions of bilirubin and two anionic drugs, warfarin and naproxen, with HSA and the three fragments. The primary bilirubin binding site on HSA molecule appeared to be located between fragment A and fragment C. The results also suggest the binding sites of the two anionic drugs to mosy likely be located in fragment C of HSA molecule.