Members of the p24 family of putative cargo receptors (subdivided into p24-alpha, -beta, -gamma and -delta) are localized in the intermediate-and cis-Golgi compartments of the early secretory pathway, and are thought to play an important role in protein transport. In the present study, we wondered what effect increased biosynthetic cell activity with resulting high levels of protein transport would have on the subcellular localization of p24. We examined p24 localization in Xenopus intermediate pituitary melanotrope cells, which in black- and white-adapted animals are biosynthetically highly active and virtually inactive respectively. In addition, p24 localization was studied in Xenopus anterior pituitary cells whose activity is not changed during background adaptation. Using organelle fractionation, we found that in the inactive melanotropes and moderately active anterior pituitary cells of white-adapted animals, the p24-alpha, -beta, -gamma and -delta proteins are all located in the Golgi compartment. In the highly active melanotropes, but not in the anterior cells of black-adapted animals, the steady-state distribution of all four p24 members changed towards the intermediate compartment and subdomains of the endoplasmic reticulum (ER), most probably the ER exit sites. In the active melanotropes, the major cargo protein pro-opiomelanocortin was mostly localized to ER subdomains and partially co-localized with the p24 proteins. Furthermore, in the active cells, in vitro blocking of protein biosynthesis by cycloheximide or dispersion of the Golgi complex by brefeldin A led to a redistribution of the p24 proteins, indicating their involvement in ER-to-Golgi protein transport and extensive cycling in the early secretory pathway. We conclude that the subcellular localization of p24 proteins is dynamic and depends on the biosynthetic activity of the cell.