A congeneric series of four bis-benzamidine inhibitors sharing a dianhydrosugar isosorbide scaffold in common has been studied by crystal structure analysis and enzyme kinetics with respect to their binding to trypsin and factor Xa. Within the series, aromatic interactions are an important determinant for selectivity discrimination among both serine proteases. To study the selectivity-determining features in detail, we used trypsin mutants in which the original binding site is gradually substituted to finally resemble the factor Xa binding pocket. The influence of these mutations has been analyzed on the binding of the closely related inhibitors. We present the crystal structures of the inhibitor complexes obtained by co-crystallizing an "intermediate" trypsin mutant. They could be determined to a resolution of up to 1.2 A, and we measured the inhibitory activity (K(i)) of each ligand against factor Xa, trypsin, and the various mutants. From these data we were able to derive a detailed structure-activity relationship which demonstrates the importance of aromatic interactions in protein-ligand recognition and their role in modulating enzyme selectivity. Pronounced preference is experienced to accommodate the benzamidine anchor with meta topology in the S(1) specificity pocket. One ligand possessing only para topology deviates strongly from the other members of the series and adopts a distinct binding mode addressing the S(1)' site instead of the distal S(3)/S(4) binding pocket.