The effect of chloroform on the chiroselective reaction between bilirubin (BR) and bovine serum albumin (BSA) at the interface between a heptane phase (including CHCl3) and an aqueous phase was investigated by means of the absorption and circular dichroism (CD) spectroscopies combined with a centrifugal liquid membrane (CLM) method, and a CLIVI microscopic fluorescence spectroscopy as well. The observed absorption, CID and fluorescence spectra disclosed the interfacial complexation process of BR with BSA for the first time, suggesting further aggregation of the BR-BSA complex at the interface. It was noticed more that, due to the formation of the chiral aggregates of BR-BSA complex, the interfacial CID signal of M(-) conformation of BR was appeared gradually. However, higher content of CHCl3 in the organic phase, resulting in the increase in fluorescence intensity, evidently affected the formation of the aggregates of the complex at the interface. The addition of extra CHCl3 to the interfacial aggregates induced temporal inversion of CID sign of BR, which should be caused by the local structural change of BSA brought about by the specific solvation of CHCl3. (C) 2008 Elsevier Inc. All rights reserved.