Annexin 1 (ANXA1) is an important mediator of glucocorticoid action in the neuroendocrine system. As the activity of this protein in other systems is modulated by phosphorylation of its N-terminal domain, we have explored the significance of this domain and its phosphorylation status to ANXA1 actions within the pituitary gland, using an established in vitro preparation. Two N-terminal peptides, ANXA1_Ac2–26 and ANXA1_Ac1–50, inhibited forskolin-evoked ACTH and prolactin release; however, they lacked the potency and full efficacy of the parent molecule (ANXA1_1–346), whereas other shorter N-terminal sequences were without effect. A chimeric protein comprising ANXA1_1–44 and the C-terminal core of ANXA5 (ANXA5_20–320) also produced a partial inhibition of peptide release. Protein kinase C (PKC) blockade (PKC_19–36) abolished the inhibitory effects of dexamethasone on forskolin-evoked peptide release and attenuated the antisecretory actions of ANXA1_Ac2–26. ANXA5, which sequesters PKC in other systems, produced similar effects. PKC_19–36 also blocked the dexamethasone- induced translocation of a serine phosphorylated species of ANXA1 from the cytoplasm to the outer cell surface. These results suggest that 1) the N-terminal domain plays a fundamental role in effecting the inhibitory actions of ANXA1 on pituitary peptide release; 2) PKC-dependent mechanisms are essential for both the cellular exportation and the biological activity of ANXA1; and 3) ANXA1 exported from the cells is serine phosphorylated.