Prohormone convertase 2 (PC2) functions in the generation of neuropeptides from their precursors. A quantitative peptidomics approach was used to evaluate the role of PC2 in the processing of peptides in a variety of brain regions. Altogether, 115 neuropeptides or other peptides derived from secretory pathway proteins were identified. These peptides arise from 28 distinct secretory pathway proteins, including proenkephalin, proopiomelanocortin, prodynorphin, protachykinin A and B, procholecystokinin, and many others. Forty one of the peptides found in wild-type (WT) mice were not detectable in any of the brain regions of PC2 knockout mice, and another 24 peptides were present at levels ranging from 20% to 79% of WT levels. Most of the other peptides were not substantially affected by the mutation, with levels ranging from 80% to 120% of WT levels, and only three peptides were found to increase in one or more brain regions of PC2 knockout mice. Taken together, these results are consistent with a broad role for PC2 in neuropeptide processing, but with functional redundancy for many of the cleavages. Comparison of the cleavage sites affected by the absence of PC2 confirms previous suggestions that sequences with a Trp, Tyr, and/or Pro in the P1' or P2' position are preferentially cleaved by PC2 and not by other enzymes present in the secretory pathway.