The effects of adrenalectomy (ADX) on the vesicular zinc content of zinc-enriched (ZEN) terminals in mouse hippocampus were investigated at light microscopic levels using zinc transporter-3 immunohistochemistry (ZnT3 IHC ) and zinc selenium autometallography (ZnSe AMG ). ZnT3 resides in the synaptic vesicle membranes of ZEN neurons and is believed to move zinc ions into the vesicles. ZnT3 IHC staining closely corresponds to the ZnSe AMG staining, but in the present study we present evidence of a delayed decrease of ZEN zinc, as compared to downregulation of the ZnT3 protein following ADX. Twenty-four hours after adrenalectomy the level of ZnT3 IHC was visibly reduced while the ZnSe AMG staining intensity seemed unchanged. After 10 and 30 days, however, downregulation of ZnT3 was paralleled by a distinct reduction in ZnSe AMG staining. The total protein concentration of ZnT3 was reduced by about 53%, and the total zinc concentration in the hippocampus of the same mice was reduced by 43–64%, 30 days after the adrenalectomy. The present results support previous results suggesting that ZnT3 is responsible for transport of zinc ions into a pool of synaptic vesicles in ZEN terminals.