Crystal structures of thrombin complexed with two spin labels called para-V, 4-(2,2,5,5-tetramethylpyrrolidine-1-oxyl)-p-(fluorosulfonyl) benzamidine, and meta-V, 3-(2,2,5,5-tetramethyl-pyrrolidine1-oxyl)-m-(fluorosulfonyl) benzamidine, have been completed at 2.0 and 3.0 A resolution, respectively. Previous electron spin resonance studies with these labels gave rise to a low-resolution "topography map" of thrombin's extended active site. These labels monitor two distinct areas of the thrombin active site: (1) an apolar binding site which manifests itself in an biphasic activation/inhibition effect on thrombin activity and (2) a region sensitive to alpha-thrombin autoproteolytic cleavage(s) to gamma-thrombin (Arg75-Tyr76 and/or Arg77A-Asn78, and Lys149E-Gly150, chymotrypsin numbering). Para-V was found to bind along the substrate binding cleft, while meta-V was found to bind both at the substrate primary specificity pocket and at a site which interacts with the gamma-cleavage loop. These studies reaffirm that accurate information may be gained from solution studies and indicates the complementarity of solid-state studies.