The hormone-binding domain of the glucocorticoid receptor must be bound to heat shock protein (hsp) 90 for it to have a high-affinity steroid binding conformation. We have recently demonstrated that hsp70 is required for cell-free assembly of the receptor.hsp90 complex and concomitant activation of steroid binding activity (Hutchison, K.A., Dittmar, K.D., Czar, M.J., and Pratt, W. B. (1994) J. Biol. Chem. 269, 5043-5049). hsp90 and hsp70 are known to exist together in a cytosolic complex containing several other proteins, and in this work we ask if all of the factors required for proper receptor folding and heterocomplex assembly are preassociated in this multiprotein complex. The multiprotein complex was immunoadsorbed to protein A-agarose from rabbit reticulocyte lysate using the 3G3 monoclonal IgM directed against hsp90. When this immunopellet is mixed with immunadsorbed mouse glucocorticoid receptor and incubated at 30 degrees C with ATP/Mg2+ and KCl, the receptor is converted to the steroid binding conformation. When the immunoadsorbed multiprotein hsp90 complex is washed extensively, it loses a weakly bound protein (not hsp70 or hsp90) that is required for receptor activation. This protein factor is contained in a Centricon C-100 filtrate of lysate which reconstitutes the receptor activating activity of the washed hsp90 complex. The hsp90 complex can be released from the 3G3 antibody, and in the presence of the protein factor in the Centricon C-100 filtrate it converts the receptor into a functional heterocomplex with hsp90. The results support the proposal that the various components of reticulocyte lysate that are required to refold the glucocorticoid receptor to the steroid binding state are preassociated with each other, acting as a self-sufficient protein folding machine.