The contribution of an extended interaction site in tissue kallikreins to their substrate specificity was investigated using peptides of increasing length and with different amino acids in positions P5 and P6. These substrates were constructed from a consensus dodecapeptide sequence (VASPFRSYDLDA) deduced from the hydrolysis of short synthetic peptide substrates, and from the identification of the cleavage sites in reduced-pyridylethylated lysozyme by 6 rat tissue kallikreins. Though the specificity constant kcat/Km generally increases with increasing the peptide substrate length on its N-terminal end, individual residues at P4-P6 may specifically alter this value for specific kallikreins. A seryl residue at P4 induces a 20-fold decrease in the specificity constant with rK2 and rK9, but it slightly improves this value for rK1 and rK10. A tryptophan in P6 is unfavourable for both rK1 and rK2 but not for rK9 and rK10, whereas a negatively charged residue has a negative effect for all four kallikreins. This demonstrates the importance of an extended interaction site in kallikreins, and suggests that the differing specificities of individual kallikreins are partly due to the presence of proteinase subsites which accommodate residues remote from the scissile bond in the substrate. These sites could be located in variable loops that surround the kallikrein active sites, and correspond to regions of lower structural similarity. Molecular modeling studies indicate that loop 4 may contribute to the P4-P7 specificity of kallikreins.