Trypsin and thrombin, structurally similar serine proteases, recognize different substrates; thrombin cleaves after Arg whereas trypsin after Lys/Arg. Both recognize basic substrate headgroups via Asp189 at the bottom of the S1-pocket. By crystallography and ITC, we studied a series of D-Phe/-D-DiPhe-Pro-(amino)pyridines. Identical ligand pairs show the same binding poses. Surprisingly, one ligand binds to trypsin in protonated, to thrombin in unprotonated state at P1 along with differences in the residual solvation pattern. While trypsin binding is mediated by an ordered water molecule, in thrombin water is scattered over three hydration sites. Although having highly similar S1-pockets, our results suggest different electrostatic properties of Asp189 possibly contributing to the selectivity determinant. Thrombin binds a specific Na+-ion next to Asp189 which is absent in trypsin. The electrostatic properties across the S1-pocket are further attenuated by charged Glu192 at the rim of S1 in thrombin which is replaced by uncharged Gln192 in trypsin.