Quantitative peptidomics was used to compare levels of peptides in wild type (WT) and Cpe(fat/fat) mice, which lack carboxypeptidase E (CPE) activity because of a point mutation. Six different brain regions were analyzed: amygdala, hippocampus, hypothalamus, prefrontal cortex, striatum, and thalamus. Altogether, 111 neuropeptides or other peptides derived from secretory pathway proteins were identified in WT mouse brain extracts by tandem mass spectrometry, and another 47 peptides were tentatively identified based on mass and other criteria. Most secretory pathway peptides were much lower in Cpe(fat/fat) mouse brain, relative to WT mouse brain, indicating that CPE plays a major role in their biosynthesis. Other peptides were only partially reduced in the Cpe(fat/fat) mice, indicating that another enzyme (presumably carboxypeptidase D) contributes to their biosynthesis. Approximately 10% of the secretory pathway peptides were present in the Cpe(fat/fat) mouse brain at levels similar to those in WT mouse brain. Many peptides were greatly elevated in the Cpe(fat/fat) mice; these peptide processing intermediates with C-terminal Lys and/or Arg were generally not detectable in WT mice. Taken together, these results indicate that CPE contributes, either directly or indirectly, to the production of the majority of neuropeptides.