We examined the effect of brefeldin A (BFA), a drug that inhibits the intracellular translocation of newly synthesized glycoproteins, on the biosynthesis of TSH and free alpha-subunits by pituitary tissue from hypothyroid mice. Incubation of tissue with 5 or 10 micrograms BFA/ml for 3.5 h caused marked dilatation of rough endoplasmic reticulum (RER) and mild swelling of Golgi in all pituitary cell types. As judged by incorporation of [35S]Met into acid-insoluble radioactivity, BFA at a concentration of 5 micrograms/ml did not substantially inhibit protein synthesis, but markedly reduced protein secretion. After a 2-h pulse with [35S]Met, followed by a 4-h chase, BFA at 5 micrograms/ml reduced the release of TSH and free alpha-subunits into the medium by 94% and 99%, respectively; subunits that accumulated within cells were forms with mol wt 2000-4000 less than normal. BFA also partially inhibited the release into the medium of TSH or free alpha-subunits labeled with [3H]fucose or [35S]SO4, but this effect was less marked than that for [35S]Met-labeled subunits. Both the morphological and the isotopic data suggest that BFA blocks transport of secretory proteins between rough endoplasmic reticulum and Golgi of pituitary cells, although transport within the Golgi may also be affected to some extent.