As an initial step toward investigating whether CT variants with five and one native GM 1 BS trafficked from the cell surface to the ER by the same pathway, we examined the effects of brefeldin A (BFA) on their toxicity for T84 cell monolayers. We found that BFA completely inhibited the I sc (Fig. 5) induced by both single and five native GM 1 BS toxin variants. BFA-treated cells, however, still responded to addition of the cAMP agonist vasoactive intestinal polypeptide (VIP) at 90 min, showing that the toxin-treated cells retained viability and were competent for cAMP-dependent Clsecretion (I sc ). Thus, a toxin variant with a single native GM 1 BS, like native CT holotoxin, traffics through a BFA-sensitive pathway to exert its toxic effects.