The intracellular pathway following receptor-mediated endocytosis of cholera toxin was studied using brefeldin A (BFA), which inhibited protein secretion and induced dramatic morphological changes in the Golgi region. In both mouse Y1 adrcnal cells and CHO cells, BFA at 1 pgiml caused a 80-90% inhibition of the cholera toxin (CT)-induced elevation of intracellular CAMP. The inhibition of the cytntoxicity of CT by BFA was also observed i n a rounding assay of Y1 adrenal cells. The inhibition of CT cytotoxicity by BFA was dose dependent, with the I D, , value similar to the LD, , of BFA in Y1 adrenal cells. Binding and internalization of ['L'I]-labeled cholera toxin in Y1 adrenal cells was not affected by BFA. Unlike the BFA-sensitive cell lines such as Y1 adrenal and CHO cells, BFA at 1 pgiml did not inhibit the cytotoxicity of CT in PtK, cells, of which the Golgi structure was BFA-resistant. These results strongly suggest that a BFA-sensitive Golgi is required for the protection of CT cytotoxicity by BFA. In contrast, elevation of the intracellular CAMP by forskolin, which acts directly on the plasma membrane adenylate cyclasc, was not affected by BFA. These observations indicate that the intoxication of target cells by CT requires an intact Golgi region for its intracellular trafficking and/or processing. In this respect, CT shares a common intracellular pathway with ricin, fseudomonas toxin, and modeccin, even though their structures and modes of action are very different.