Prohormone convertase 2 (PC2) requires interaction with the neuroendocrine protein 7B2 for the production of an activatable zymogen; the mechanism for this effect is unknown. 7B2 could act proactively to generate an activation-competent form of proPC2 during synthesis, or block spontaneous generation of activation-incompetent forms. We here demonstrate that addition of exogenous recombinant 7B2 to CHO cells expressing proPC2 prevented the unfolding and aggregation of secreted PC2 forms in a dose-dependent manner, as assessed by aggregation assays, activity assays, cross-linking experiments, and sucrose density gradients. Intracellular proPC2 was also found to exist in part as higher-order oligomers which were reduced in the presence of co-expressed 7B2. 7B2 addition did not result in the acquisition of enzymatic competence unless added prior to or very rapidly after proPC2 secretion, indicating that an activation-competent structure cannot be maintained in the absence of 7B2. Velocity sedimentation experiments showed that addition of extracellular 7B2 solubilized three different PC2 species from a precipitable aggregate: two activatable proPC2 species, the intact zymogen and a zymogen with a partially cleaved propeptide; and an inactive 66 kDa form. Our results suggest that 7B2 possesses chaperone activity which blocks partially unfolded proPC2 forms from losing catalytic competence and then aggregating. The loss of the catalytically competent conformer appears to represent the earliest indicator of proPC2 unfolding and is followed on a slower time scale by the appearance of aggregates. Since 7B2 expression is not confined to areas expressing proPC2, 7B2 may represent a general intracellular and extracellular secretory chaperone.