The binding of two cholecystographic agents to human serum albumin (HSA) was evaluated by means of two different complementary methodologies. In particular, the inhibition of drug HSA binding caused by iopanoic- and iophenoxic-acid was investigated by circular dichroism (CD) and resonant mirror (RM) optical biosensor techniques. The CD study allowed to obtain information both on the cholecystographic agent binding site and on the effect of the binding on the protein conformation. Iopanoic acid (IOP), a drug potentially useful for thyrotoxic disorders, resulted a direct competitor for ligands that selectively bind to site II, in agreement to literature data. No definite evidence was obtained for the highest affinity binding site of iophenoxic acid (IOPH), however, this diagnostic tool markedly affected the binding of ligands to the most characterized high affinity sites on HSA, namely sites I, II and III. Binding parameters were obtained by optical biosensor analysis: K(D) values were 3.6 x 10(-7) and 2.8 x 10(-8) M for IOP and IOPH, respectively.