The effect of ammonium on protein processing and secretion from mouse insulinoma beta TC3 cells and recombinant mouse pituitary AtT-20 cells uas investigated. Protein stored in granules of the regulated secretion pathway was discharged from cells with secretagogues, and the addition of new protein was encouraged by recharging cells in serum-containing medium with or without added ammonium. Ammonium at 6 mM inhibits the addition of insulin-related peptides to intracellular stoles in both cell lines; it is not clear whether this effect is dose dependent for concentrations between 0 and 6 mM. There is a slight increase in insulin-related proteins secreted during recharging of cells in the presence of ammonium. Using reverse-phase high-performance liquid chromatography to separate proinsulin from insulin, we found that extracts from beta TC3 cells recharged in the presence of 6 mM ammonium contain significantly less insulin than control cells recharged in the absence of ammonium.