Translocation to the cytosol is an essential and rate-limiting step in the cytotoxicity of the potent plant toxin ricin. In an attempt to study the mechanism of ricin A-chain translocation in a cell-free assay, we have partially purified Golgi and endoplasmic reticulum from Jurkat cells by discontinuous sucrose gradient fractionation. The membranes of the organelle fractions were solubilized by the addition of sodium cholate and reconstituted into proteoliposomes by dialyzing out the detergent. The resulting vesicles supported cell-free translocation of RTA (as assessed by an enzyme protection assay) at a rate which was linearly dependent on the concentration of the vesicle preparation. Ricin B-chain (RTB) neither translocated into the vesicles, nor increased the efficiency of RTA translocation. Liposomes prepared from purified phospholipids were not capable of supporting RTA translocation. Furthermore, protease treatment of concanavalin A adsorption of proteins from lysates prior to vesicle reconstitution resulted in abrogation of the translocation process, suggesting that the protein components of organelle membranes are required for RTA translocation. Reconstitution of translocation-competent proteoliposomes from detergent-solubilized membranes of endoplasmic reticulum- and Golgi-enriched fractions provides a convenient cell-free system to study the mechanism of RTA translocation.