Structure-function relationships in the 6 epidermal growth factor-like domains of human thrombomodulin (TME, residues 227-462) were studied by deletion mutagenesis. Purified and characterised proteins were used for kinetic studies. Deletion of EGF1, EGF2 and residues 310-332 in EGF3 had no effect on thrombin binding (Kd) or on kcat/KM for protein C activation by the thrombin-thrombomodulin complex. Deletion of the rest of EGF3 and the interdomain loop between EGF3 and EGF4 had no effect on Kd but decreased kcat/KM to 10% of TME. Deletion of residues 447-462 of EGF6 had no effect on kcat/KM but increased Kd for thrombin approximately 6-fold. Thus, the region 333-350 in EGF3-4 is critical for protein C activation by the thrombin-thrombomodulin complex and the region 447-462 in EGF6 is critical for thrombin binding.