ABSTRACT The active pool of internalized cholera toxin (CT) moves from the endosomes to the Golgi apparatus en route to the endoplasmic reticulum (ER). The catalytic CTA1 polypeptide is then translocated from the ER to the cytosol, possibly through the action of the ER-associated degradation (ERAD) pathway. Translocation was previously measured indirectly through the downstream effects of CT action. We have developed a direct biochemical assay for CTA1 translocation that is independent of toxin activity. Our assay is based upon the farnesylation of a CVIM motif-tagged CTA1 polypeptide (CTA1-CVIM) after it enters the cytosol. When expressed from a eukaryotic vector in transfected CHO cells, CTA1-CVIM was targeted to the ER, but was not secreted. Instead, it was translocated into the cytosol and degraded in a proteosome-dependent manner. Translocation occurred rapidly and was monitored by the appearance of farnesylated CTA1-CVIM in the detergent phase of cell extracts generated with Triton X-114. Detergent-phase partitioning of CTA1-CVIM resulted from the cytoplasmic addition of a 15-carbon fatty acid farnesyl moiety to the cysteine residue of the CVIM motif. Our use of the CTA1-CVIM translocation assay provided supporting evidence for the ERAD model of toxin translocation and generated new information on the timing of CTA1 translocation.