Ricin translocation was demonstrated (using both fluorescence- and radiolabel-based assays) across the membrane of endosomes purified from mouse lymphocytes. Selectivity of the process was shown by the absence of translocation activity of transferrin and horseradish peroxidase used as membrane-bound and fluid-phase endosome labels, respectively. Endocytosed 125I-ricin translocation was found to be strictly ATP- (Km approximately 4 mM) and temperature-dependent, with up to 30% endosomal 125I-ricin appearing in the external medium after 2 h at 37 degrees C. No treatments neutralizing the acidic endosome pH (ammonium chloride, nigericin, chloroquine) significantly impaired ricin translocation, and the pH gradient across the endosome membrane is not required for this process. Chase experiments showed that the ability of 125I-ricin to translocate increases with its depth in the endocytic system (i.e. plasma membrane << early endosomes < late endosomes). Both A and B ricin chains displayed translocation ability as demonstrated by the results of our assay on ricin, ricin B, transferrin-ricin A, and transferrin-ricin B conjugates. Biological activity of both ricin chains is preserved after translocation as shown by the inhibitory effect of the A chain on cell-free protein synthesis and the binding of the B chain to lactose-agarose.