The interaction of bilirubin with de‐fatted human serum albumin has been studied by circular dichroism and spectrofluorimetry. From the dependence of the induced circular dichroism of the bilirubin on the ratio of protein to bilirubin, it is shown that in the presence of salt (0.5 M sodium chloride) there are two binding sites with markedly different optical characteristics. From an analysis of the profiles of ellipticity as a function of protein: bilirubin ratio, association constants of 7 and 0.3 μM−1 at pH 8.5 were derived. At low salt concentrations qualitatively similar binding profiles are obtained with a marked maximum of ellipticity at a protein: bilirubin ratio of about 1. These cannot however be adequately accounted for with only two binding sites. The addition of a third site allows the data to be fitted over the entire concentration range at all wavelengths tested, with association constants falling in the range 10–0.01 μM−1. When bilirubin is bound to albumin, the fluorescence of the protein is quenched and that of the bilirubin is enhanced. The Fluorescence characteristics of the binding sites are shown to be different: the weaker sites, which have lower fluorescence, but higher circular dichroism than the stronger, are relatively much more labile to salt. The pH‐dependence of the optical properties reveals the presence of a state defined by a plateau at between pH 8 and 9, another by a plateau centred at about pH 6.5, and another, the appearance of which is associated with the N–F transition of the protein, and which exists only within an extremely narrow pH range, at about pH 4. The pH at which the minimum occurs undergoes a large shift with salt concentration. This state is characterised by an extremely large circular dichroism (effective molar ellipticity ‐5 × 105 deg. cm2· dmol−1), opposite in sign to the system of Cotton effects observed at higher pH. This complex shows no fluorescence enhancement. The system shows large hysteresis, satisfactory results being obtained only when the complex is first formed by equilibration at pH 8–9 at low ionic strength. Under other conditions the self‐association of the bilirubin evidently prevents the attainment of equilibrium, and vitiates any attempts at analysis of the binding equilibria. The competition of oleate ions with the bilirubin binding sites has been studied. Circular dichroism, under physiological conditions, of human serum containing bilirubin shows that the binding of the bilirubin by albumin alone at the corresponding ionic strength and pH defines the state of the ligand in the plasma.