Forskolin has been shown to prevent the effects brefeldin A (BFA) exerts on many mammalian cells with respect to the disassembly of the Golgi apparatus as well as an increase of sphingomyelin synthesis (Lippincott, S. J., Glickman, J., Donaldson, J. G., Robbins, J., Kreis, T. E., Seamon, K. B., Sheetz, M. P., and Klausner, R. D. (1991) J. Cell Biol. 112, 567-577). It has been speculated that forskolin interferes with the action of BFA by competition for the binding of BFA to its target protein, which is most likely the Golgi-localized nucleotide exchange factor specific for ADP-ribosylation factor 1. Here we show that in vitro forskolin does not prevent inhibition of Golgi-catalyzed nucleotide exchange by BFA. Therefore it appears unlikely that forskolin and BFA bind to the same target protein. Using [3H]BFA we have measured detoxification of BFA by Chinese hamster ovary (CHO) cells. BFA is secreted from CHO cells as cysteine and glutathione conjugates (Brüning, A., Ishikawa, T., Kneusel, R. E., Matern, U., Lottspeich, F., and Wieland, F. T. (1992) J. Biol. Chem. 267, 7726-7732). We present evidence that forskolin treatment of CHO cells results in increased levels of Cys-BFA, the major BFA conjugate secreted by CHO cells, in the medium. Elevated levels of Cys-BFA are also found intracellularly. The effect of forskolin is shown to be independent of its ability to raise the intracellular concentration of cyclic AMP. Therefore, we suggest that the effect of forskolin on BFA-induced disassembly of the Golgi apparatus might be due to an enhanced detoxification of the drug.