We have found that brefeldin A (BFA) inhibited the growth of an isel mutant of Saccharomyces cerevisiae. Genetic complementation and mapping studies demonstrated that isel was allelic to erg6, a gene required for the biosynthesis of the principal membrane sterol of yeast, ergosterol. Treatment of isel cells with BFA resulted in an immediate block in protein transport through the secretory pathway. Vacuolar carboxypeptidase Y (CPY) and the secreted pheromone a-factor accumulated as both the core glycosylated (ER) and al,6 mannosylated (early Golgi) forms in drugtreated cells. The modification of a-factor with al,6 mannose in BFA-treated cells did not appear to result from retrograde transport of the al,6 mannosyltransferase into the ER. We found that transport of CPY from medial and late Golgi compartments to the vacuole was unaffected by BFA, nor was secretion of al,3 mannosylated a-factor or invertase blocked by BFA. The effects of BFA on the secretory pathway were also reversible after brief exposure (<40 min) to the drug. We suggest that the primary effect of BFA in S.cerevisiae is restricted to the ER and the a(1,6 mannosyltransferase compartment of the Golgi complex.