Adenosine triphosphate (ATP)-affinity chromatography has been widely used to purify molecules of the Hsp70 family. This procedure leads to dissociation of peptides from Hsp70 molecules, resulting in Hsp70 preparations devoid of immunological activity. We report that substitution of ATP-affinity chromatography by ADP-affinity chromatography results in isolation of Hsp70 molecules which are still associated with peptides and are immunogenic. The Hsp70 preparations thus obtained contain the constitutive Hsp73 and the inducible Hsp72 molecules. These observations furnish a basis for an analysis of the structural heterogeneity among the members of the Hsp70 family and of the antigenic peptides associated with individual members of this family. They also provide a practical method for the isolation of large quantities of immunologically active Hsp70-peptide preparations.