Cholera toxin (CT) moves from the plasma membrane to the endoplasmic reticulum (ER) by retrograde vesicular traffic. In the ER, the catalytic CTA1 polypeptide dissociates from the rest of the toxin and enters the cytosol by a process that involves the quality control mechanism of ER-associated degradation (ERAD). The cytosolic CTA1 then ADP ribosylates Gsalpha, resulting in adenylate cyclase activation and intoxication of the target cell. It is hypothesized that the C-terminal A1(3) subdomain of CTA1 plays two crucial roles in the intoxication process: (i) it contains a hydrophobic domain that triggers the ERAD mechanism and (ii) it facilitates interaction with the cytosolic ADP-ribosylation factors (ARFs) that serve as allosteric activators of CTA1. In this study, we examined the role(s) of the CTA1(3) subdomain in CT intoxication. Full-length CTA1 constructs and truncated CTA1 constructs lacking the A1(3) subdomain were generated and used to conduct two-hybrid studies of interactions with ARF6, in vitro enzyme assays, in vivo toxicity assays, and in vivo processing/degradation assays. Direct, plasmid-mediated expression of CTA1 constructs in the ER or cytosol of transfected CHO cells was used to perform the in vivo assays. With these methods, we found that the A1(3) subdomain of CTA1 is important both for interaction with ARF6 and for full expression of enzyme activity in vivo. Surprisingly, however, the A1(3) subdomain was not required for ERAD-mediated passage of CTA1 from the ER to the cytosol. A possible alternative trigger for CTA1 to activate the ERAD mechanism is discussed.