Inhibitors of serine proteases (serpins) are important regulators of intra and extracellular proteolytic pathways, and function by forming an irreversible complex with their substrate. Neuroserpin represents a neuroendocrine-specific serpin family member that is expressed in brain regions displaying synaptic plasticity. Here we explored the biosynthesis of endogenous neuroserpin in a neuroendocrine model system, namely the melanotrope cells of Xenopus intermediate pituitary. The biosynthetic activity of these cells can be physiologically manipulated (high and low production of the prohormone proopiomelanocortin [POMC] in black and white animals, respectively), resulting from a synaptic plasticity in innervating hypothalamic neurons. We found that neuroserpin was also differentially expressed in the Xenopus intermediate, but not anterior, pituitary with a 3-fold higher mRNA- and >30-fold higher protein expression in the active vs. the inactive melanotrope cells. Two newly synthesized glycosylated forms of the neuroserpin protein (47- and 50 kDa) were produced and secreted by the active cells. Intriguingly, neuroserpin was found in an 130-kDa SDS-stable complex in the active, but not in the inactive, melanotrope cells which correlated with the high and low POMC expression levels, respectively. In conclusion, we report on the biosynthesis of neuroserpin in a physiological context, and find that the induction of neuroserpin expression and the formation of the 130-kDa neuroserpin-containing complex are linked to neuroendocrine cell activation.