To search for the substrates, other than neurotensin, of rat brain neurolysin, a novel method of determining peptidase activity was developed using a yeast molecular display system. This is a useful and convenient method of handling homogenously pure proteins to evaluate the properties of neurolysin. The neurolysin gene was ligated to the C-terminal half of the alpha-agglutinin gene with a FLAG tag sequence and a yeast cell-surface molecular displaying plasmid was constructed. Display of neurolysin with correct folding and appropriate activity was verified by immunofluorescence staining and activity measurement of a bradykinin-related peptide. The cleavage sites of peptides were determined by high-performance liquid chromatography (HPLC) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results showed the amino acid preferences of hydrophobic, aromatic, and basic residues, which were the same as those of soluble neurolysin. Moreover, this method clearly showed the presence of two recognition motifs in neurolysin. By using these motifs, novel substrate candidates of neurolysin in rat tissues were screened, and several bioactive peptides that regulate feeding were found. We also discussed the ubiquitous distribution of neurolysin in rat tissues and the functions of substrate candidate peptides.