We recently reported that pituitary gonadotropes, major targets of circulating inhibins and activins, are also capable of synthesizing the inhibin (I) alpha- and inhibin/activin (I/A) beta B-subunits. In the present study, we examined the subcellular distribution of these subunits, with special attention given to determinating the extent to which they might be colocalized with the gonadotropins in secretory granules. Pituitaries from adult male rats were cryofixed, molecular distillation-dried, and resin-embedded. Immunogold staining methods were used to examine concurrently the distributions of an I/A subunit and FSH or LH. I/A subunits were detected only in cells that also labeled positively for a gonadotropin, and, in contrast to the gonadotropins, were sequestered almost exclusively within secretory granules. The I alpha-subunit colocalized with FSH in 31%, and with LH in 36%, of all positively stained granules. The I/A beta B-subunit was found with FSH or LH in about 25% of the granules. Approximately 52-69% of the granules contained FSH or LH alone; 7-18% were positive only for an I/A subunit, and this varied as a function of the particular gonadotropin with which costaining was carried out. Dual staining for the I alpha- and the I/A beta B-subunits indicated that at least 35% of all immunolabeled granules showed positive signals for both subunits. Coupled with methodological considerations to indicate that these estimates of the extent of colocalization are likely to be conservative, these data suggest that inhibin and activin are characteristically copackaged, and presumably coreleased, with the gonadotropins.