Completion of the Caenorhabditis elegans genome sequencing project in 1998 has provided more insight into the complexity of nematode neuropeptide signaling. Several C. elegans neuropeptide precursor genes, coding for approximately 250 peptides, have been predicted from the genomic database. One can, however, not deduce whether all these peptides are actually expressed, nor is it possible to predict all post-translational modifications. Using two dimensional nanoscale liquid chromatography combined with tandem mass spectrometry and database mining, we analyzed a mixed stage C. elegans extract. This peptidomic setup yielded 21 peptides derived from formerly predicted neuropeptide-like protein (NLP) precursors and 28 predicted FMRFamide-related peptides. In addition, we were able to sequence 11 entirely novel peptides derived from nine peptide precursors that were not predicted or identified in any way previously. Some of the identified peptides display profound sequence similarities with neuropeptides from other invertebrates, indicating that these peptides have a long evolutionary history.