Contrary to the notion that the soluble intravesicular matrix proteins of the secretory vesicles of adrenal medullary chromaffin cells freely float in the vesicle, several vesicle matrix proteins of the secretory vesicles, including chromogranins A and B, bound to the vesicle membrane at intravesicular pH (5.5) and were freed from it when the pH was raised to a near physiological pH (7.5). Estimation of the fraction of vesicle matrix proteins that might remain bound to the vesicle membrane in the vesicle suggested that the majority (> 50-80%) of chromogranins A and B, as well as several other proteins, will stay bound to the membrane in the vesicle. Comparison of the amino-acid sequences of chromogranins A and B revealed two highly conserved regions, i.e., one near the N-terminus and the other being the C-terminal region. Since it has been demonstrated with chromogranin A that the conserved near N-terminal region of chromogranin A exhibited the pH-dependent membrane-binding activity (Yoo, S. H. (1993) Biophys. J., 64, A195), the same region in chromogranin B (residues 17-36) was tested using a synthetic chromogranin B peptide, and found to exhibit the pH-dependent membrane-binding activity. The pH-dependent binding of the matrix proteins at pH 5.5 and the automatic untethering at a physiological pH accord well with the rapid release and circulation of the vesicular contents in the bloodstream.