Abstract. Proinsulin is a single polypeptide chain composed of the B and A subunits of insulin joined by the C-peptide region. Proinsulin is converted to insulin during the maturation of secretory vesicles by the action of two proteases and conversion is inhibited by ionophores that disrupted intracellular H ÷ gradients. To determine if conversion of prohormone to hormone actually occurs in an acidic secretory vesicle, cultured rat islet cells were incubated in the presence of 3-(2,4-dinitroanilino)-3' amino-N-methyldipropylamine (DAMP), a basic congener of dinitrophenol that concentrates in acidic compartments and is retained there after aldehyde fixation. The cells were processed for indirect protein A-gold colocalization of DAMP, using a monoclonal antibody to dinitrophenol, and proinsulin, using a monoclonal antibody that exclusively reacts with the prohormone. The average density of DAMP-specific gold particles in immature secretory vesicles that contained proinsulin was 71/~tm: (18 times cytoplasmic background), which indicated that this compartment was acidic. However, the density of DAMP-specific gold particles in the insulin-rich mature secretory vesicle averaged 433/~tm 2. This suggests that although proinsulin conversion occurs in an acidic compartment, the secretory vesicles become more acidic as they mature. Since the concentration of anti-proinsulin IgG binding in secretory vesicles is inversely proportional to the conversion of proinsulin to insulin, we were able to determine that maturing secretory vesicles had to reach a critical pH before proinsulin conversion occurred.URING endocytosis and exocytosis, macromolecules are sequestered in a series of membrane-bound vesicles that migrate to specific intracellular targets. A remarkable feature of these vesicles is that often their interior is maintained at a low pH relative to the surrounding cytoplasm (25,28). Whereas the pH of vesicles in the endocytic pathway has been measured in situ with pH-sensitive dyes (19,35), vesicles in the secretory pathway have been isolated and the pH measured directly (4, 10, 11, 40). The pH of these vesicles ranges from pH 4.5 (19) to pH 6.5 (38, 39) depending on the type of vesicle, and there is good evidence that the proton gradient is generated by an ATP-dependent proton pump. Coated vesicles (7,8,34), endosomes (9), and lysosomes (20, 30) in the endocytic pathway and the Golgi apparatus (10, 40) and secretory vesicles (4, 11,29) in the exocytic pathway contain a similar type of proton pump. Thus, acidification of these compartments is an active process that may be regulated in response to specific stimuli.The function of low pH in these compartments is not entirely understood. When the intraluminal pH of the vesicles is neutralized, either with ionophores or basic amines, several important reactions in both the endocytic and exocytic pathways are inhibited. These include (a) receptor recycling during receptor-mediated endocytosis (3); (b) degradation of macromolecules in the lysosome (37); (c) sorting of macromol...