Ribonuclease T1 was crystallized in the presence of vanadate(V). The crystal structure was solved by molecular replacement and refined by least-squares methods using stereochemical restraints. The refinement was based on data between 10 and 1.8 A and converged at a crystallographic R factor of 0.137. Except for the substrate-recognition site the three-dimensional structure of ribonuclease T1 closely resembles the structure of the enzyme complexed with guanosine 2'-phosphate and its derivatives. A tetrahedral anion was found at the catalytic site and identified as H2VO4-. This is the first crystal structure of ribonuclease T1 determined in the absence of bound substrate analogue. Distinct structural differences between guanosine-free and complexed ribonuclease T1 are observed at the base-recognition site: The side chains of Tyr45 and Glu46 and the region around Asn98 changed their conformations, and the peptide bond between Asn43 and Asn44 has turned around by 140 degrees. We suggest that the structural differences seen in the crystal structures of free and complexed ribonuclease T1 are related to conformational adjustments associated with the substrate binding process.