The heterogeneity of the glucocorticoid binding in the chicken liver cytosol, previously suggested by the results obtained with crude preparations, was confirmed using different techniques such as stepwise ammonium sulfate precipitation, hydroxyapatite chromatography and gel filtration on Sephadex G-200. The latter method permitted the separation of five glucocorticoid binding macromolecules respectively named binders S1, S2, S3, S4 and S5, according to their decreasing apparent molecular size upon gel filtration. Apparent molecular weight, binding affinity, capacity and specificity of these five moieties were examined. In addition, S-aryl-transferase activity using glutathione as co-substrate was studied and found to coincide mostly with the fractions containing binder S4, which might represent an avian liver ligandin.