The acidic environment inside secretory vesicles ensures that neuropeptides and peptide hormones are packaged in a concentrated condensed form. Although this is optimal for storage, decondensation limits release. Thus, it would be advantageous to alter the physical state of peptides in preparation for exocytosis. Here, we report that depolarization of the plasma membrane rapidly increases enhanced green fluorescent protein (EGFP)-tagged hormone fluorescence inside secretory vesicles. This effect requires Ca²⁺ influx and persists when exocytosis is inhibited byN-ethylmaleimide. Peptide deprotonation appears to produce this response, because it is not seen when the vesicle pH gradient is collapsed or when a pH-insensitive GFP variant is used. These data demonstrate that Ca²⁺ evokes alkalinization of the inside of secretory vesicles before exocytosis. Thus, Ca²⁺ influx into the cytoplasm alters the physical state of intravesicular contents in preparation for release.