To elucidate the mechanisms of cuproprotein biosynthesis in the secretory pathway, a polyclonal antiserum was generated against hephaestin, a multicopper oxidase essential for enteric iron absorption. Immunoblot analysis and pulse-chase metabolic labeling revealed that hephaestin is synthesized as a single-chain polypeptide modified by N-linked glycosylation to a mature 161-kDa species. Cell surface biotinylation and immunofluorescent studies of polarized, differentiated colon carcinoma cells detected hephaestin on the basolateral surface under steady-state conditions. However, a decrease in the intracellular copper concentration resulted in a marked diminution in the abundance of this protein. Metabolic studies revealed no effect of decreased intracellular copper on the rate of hephaestin synthesis but a dramatic, specific, and reproducible increase in the turnover of the mature 161-kDa protein. Surprisingly, inhibitor studies revealed that this turnover occurs exclusively in the proteasome, and consistent with this finding, in vitro studies identified polyubiquitinated hephaestin under conditions abrogating copper incorporation into this protein. Taken together, these studies demonstrate the presence of a quality control system for posttranslational protein modification occurring beyond the endoplasmic reticulum that, in the case of hephaestin, directly links the rate of enteric iron uptake to nutritional copper status.