Recombinant human adenovirus serotype 2 proteinase (both native and selenomethionine-substituted) has been crystallized in the presence of the serotype 12, 11-residue peptide cofactor. The crystals (space group P3121 or P3221, one molecule per asymmetric unit, a = b = 41.3 Å, c = 197.0 Å) grew in solutions containing 20–40% 2-methyl-2,4-pentanediol (MPD), 0.1–0.2 M sodium citrate, and 0.1 M sodium HEPES, pH 5.0–7.5. Diffraction data (84% complete to 2.2 Å resolution with Rmerge of 0.0335) have been measured from cryopreserved native enzyme crystals with the Argonne blue (1,024 × 1,024 pixel array) charge-coupled device detector at beamline X8C at the National Synchrotron Light Source (operated by Argonne National Laboratory's Structural Biology Center). Additionally, diffraction data from selenomethionine-substituted proteinase, 65% complete to 2.0 Å resolution with Rmerge values ranging 0.05–0.07, have been collected at three X-ray energies at and near the selenium absorption edge. We have determined three of the six selenium sites and are initiating a structure solution by the method of multiwavelength anomalous diffraction phasing.