In rat and mouse the phylogenetic homologues of the human mast cell α-chymase (rMCP-5 and mMCP-5) have lost their chymase activity and instead become elastases. To investigate whether rodents hold enzymes with equivalent function as the primate α-chymases, we have determined the extended cleavage specificity of the major connective tissue mast cell β-chymases in rat and mouse, rMCP-1 and mMCP-4. By using a phage display approach we determined the enzyme/substrate interaction in seven positions, both N- and C-terminal of the cleaved bond. The two proteases were found to display rather similar specificities. Both enzymes prefer Phe in position P1, and aliphatic amino acids are favoured N-terminal of the cleaved bond, i.e. Leu in P2 and Val in P3 and P4. Val and Leu are overrepresented also in positions P1′ and P3′. The two enzymes differ clearly only in one position, the P2′ residue, where mMCP-4 strongly prefers negatively charged amino acids while rMCP-1 favours Ser. Interestingly, Asp and Glu are often present in position P2′ of known substrates for the human chymase. Overall, these two rodent β-chymases have very similar amino acid preferences as the human chymase, particularly mMCP-4, which most likely have a very similar function as the human chymase. This finding indicates that rodent and primate connective tissue mast cells seem to have relatively similar proteolytic repertoires, although they express different sets of serine proteases.