Cholera toxin enters cells via an unusual pathway that involves trafficking through endosomes to the endoplasmic reticulum (ER). Whether the toxin induces its own pathway or travels along a physiological retrograde route is not known. To study its trafficking, we labeled cholera toxin B (CTB) or endogenous plasma membrane proteins with a small chemical compound, benzylguanine, which covalently reacts with the protein SNAP-tag. Using ER-targeted SNAP-tag as reporter, we found that transport of CTB to the ER depends on dynamin-2 and syntaxin 5. Plasma membrane proteins and a fluid-phase marker added to the medium were also transported to the ER. This flux was not affected by exposing cells to CTB but was inhibited by depleting syntaxin 5 and increased by depleting dynamin-2. As a control for confined intracellular localization of ER-targeted SNAP-tag we used adenovirus-5, which traffics to endosomes and then escapes into the cytosol. The virus did not react with ER-targeted SNAP but with cytosolic SNAP. Together, our results establish a new method (SNAP-trap) to study trafficking of different cargo to the ER and the cytosol and provide evidence for the existence of a constitutive pathway from the cell surface to the ER.