The rat kallikrein rK9 is one of the six members of the rat tissue kallikrein family isolated to date. It is 84% identical to rK2 (tonin), and both proteinases are thought to have vasoconstrictive properties. Recently we have shown that rK9 and rK2 have distinct substrate specificities and sensitivities to inhibitors, despite their similar sequences. Unlike all other mammalian kallikrein-related proteinases, rK9 is resistant to inhibition by aprotinin. We have developed a 3-D model of rK9, based on the known X-ray structures of rK2, porcine kallikrein and bovine trypsin, to identify the structural features underlying this functional diversity. The final rK9 model is structurally similar to rK2, but variable regions surrounding the active site differ quite markedly from the reference proteins. The kallikrein loop, which differs from that in porcine kallikrein by a seven-residue insertion, has been generated de novo and subjected to simulated annealing to assess its influence on the restricted substrate specificity of these proteinases. The proposed conformation of the specificity pocket in rK9 differs from that of other serine proteinases, but it can still accommodate both aromatic and basic amino acid side chains at the substrate P1 position, thus explaining the dual chymotrypsin and trypsin-like activity of rK9. The electrostatic potentials of rK9 and aprotinin were calculated using the finite difference Poisson-Boltzmann method. They indicated a large positive region near the active site of rK9 not found in related proteinases because of positively charged residues at positions 61 and 65 in rK9. They generate a positive region, which overlaps a positive region in aprotinin, and may prevent aprotinin binding. A single mutation in aprotinin is suggested that might allow kallikrein rK9 inhibition by aprotinin. This model contributes significantly to our understanding of the structure-function relationships among proteinases of the tissue kallikrein family.