PRL in secretory granules is osmotically inert. Previous studies by us and others have suggested that this is due in part to hormone oligomerization. Data suggest intermolecular disulfide bridges and/or intermolecular ionic interactions, as thiols, urea, and chelators increase monomerization of the majority of granule PRL. Because of the inhibitory effect of zinc on PRL release from isolated granules and the effects of zinc on the specific packing of PRL within granules, we examined the possibility that zinc contributed to the stability and/or oligomerization of intragranular PRL. To do this, we first analyzed zinc binding to purified monomeric rat PRL in solution. Zinc binding was demonstrated using the chromogenic chelator 5,5'-nitrilodibarbituric acid (murexide) and was confirmed by matrix-assisted, time of flight mass analysis. Because these spectrophotometric methods were not applicable for intragranular PRL studies, we tested the influence of zinc on granule PRL indirectly. As hormone free thiols were potentially formed during PRL oligomerization and storage, these were possible sites for hormone-divalent cation interactions. By derivatization of thiols with 4-vinyl pyridine and isolation of the carboxyterminal region of granule PRL, we found that a proportion of the cysteines 189 and 197 occurred as thiols and not disulfides. These thiols were only detectable when EDTA was present in the granule incubations. It is proposed that binding of zinc stabilizes the intermolecularly bonded storage form of PRL, in part by protection of hormone free thiols. Removal of the divalent cation and exposure of free thiols could be what initiates the thiol-disulfide interchange necessary for conversion of intermolecular to intramolecular disulfide bonds before exocytosis. Experimentally, both urea and EDTA could remove zinc, thus initiating monomerization and explaining how these agents as well as thiols achieve monomerization.