SummaryPrevious studies have suggested that chromogranin A (CgA) is a partner molecule of secretogranin III (SgIII). In mouse pituitary corticotroph-derived AtT-20 cells, SgIII plays a role in sorting CgA/hormone aggregates into secretory granules (SGs). Although CgA expression is equivocal, CgB is clearly detectable in the rat pituitary corticotrophs. Therefore, we hypothesized that CgB shares a function with CgA in pituitary corticotrophs. In the binding assays, CgB, similar to CgA, showed binding activity to SgIII under weakly acidic conditions and in the presence of Ca2+. Considering the differences in animal species, the different abilities of antibodies, and the conditions of tissue fixation and thin sectioning in immunofluorescence histochemistry, we found that CgA was expressed in a small population (approximately 10%), and its expression intensity was weaker than that of CgB (>98%) in rodent pituitary corticotrophs. In addition, similar to CgA, CgB and SgIII were colocalized in adrenocorticotropic hormone (ACTH) granules. The labeling of CgA and CgB was not completely consistent, and CgB colocalized with SgIII in many granules. These results suggest that there are multiple sorting systems for ACTH granules in pituitary corticotrophs and that the SgIII/CgB complex behaves more dominantly than the SgIII/CgA complex, which has somewhat different properties.