The human chymase (HC) is a major granule constituent of mast cells (MCs) residing in the connective tissue and the sub-mucosa. Although many potential substrates have been described for this important MC enzyme, its full range of in vivo substrates has most likely not yet been identified. A major step toward a better understanding of the function of the HC is therefore to determine its extended cleavage specificity. Using a phage-displayed random nonapeptide library, we show that the HC has a rather stringent substrate recognition profile. Only aromatic amino acids (aa) are accepted in position P1, with a strong preference for Tyr and Phe over Trp. Aliphatic aa are preferred in positions P2 to P4 N-terminal of the cleaved bond. In the P1' position C-terminal of the cleaved bond, Ser is clearly over-represented and acidic aa Asp and Glu are strongly preferred in the P2' position. In P3', the small aliphatic aa Ala, Val and Gly were frequently observed. The consensus sequence, from P4 to P3': Gly/Leu/Val-Val/Ala/Leu-Ala/Val/Leu-Tyr/Phe-Ser-Asp/Glu-Ala/Val/Gly, provides an instrument for the identification of novel in vivo substrates for the HC. Interestingly, a very similar cleavage specificity was recently reported for the major chymase in mouse connective tissue mast cells (CTMCs), the beta-chymase mouse mast cell protease-4, suggesting functional homology between these two enzymes. This indicates that a rather stringent chymotryptic substrate recognition profile has been evolutionary conserved for the dominant CTMC chymase in mammals.