We have previously shown that in HeLa cells, Shiga toxin B-fragment is transported retrogradely from the plasma membrane via endosomes and the Golgi apparatus to the endoplasmic reticulum, (J. Biol. Chem., 272, 19554-19561). Here we found that during incubation at low temperatures (19.5 o C), B-fragment accumulated in cytoplasmic vesicular structures which were also labeled by cointernalized transferrin, a marker protein of early/recycling endosomes. In contrast, B-fragment colocalization with fluid phase markers was less complete, and lysosomebound epidermal growth factor was found in structures adjacent to B-fragment positive compartments. Upon shift to 37 o C, some internalized B-fragment was recycled to the cell surface, while the majority was transported to the trans-Golgi network. By confocal microscopy on living cells, the half-time of B-fragment transport from early/recycling endosomes to the Golgi apparatus was found to be 19 min. Ultrastructural studies on cryosections showed that during this transport, no B-fragment specific label appeared in multivesicular or multilamellar late endosomes. We then showed by morphological and biochemical experiments that drugs with effects on the actin cytoskeleton (cytochalasin D) and on endosomal pH (bafilomycin, weak bases) that have been found to modulate vesicular transport in the late endocytic pathway did not affect B-fragment transport to the Golgi apparatus. In contrast, brefeldin A treatment lead to the accumulation of the B-fragment in transferrin-receptor containing tubular elements. We also report that upon incubation at 19.5 o C, B-fragment and γ-adaptin colocalized on membranes of early/recycling endosomes, suggesting a role of adaptor protein type 1 clathrin coats in B-fragment exit from early/recycling endosomes. Taken together, these results are consistent with the hypothesis that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus. This transport route may not be limited to the B-fragment but could also be a pathway used by other cellular proteins.