Crystals have been obtained of the recombinant human adenovirus serotype 2 proteinase (AVP) in a complex with its 11-amino-acid cofactor pVIc. AVP-pVIc complexes were formed by the incubation of AVP with a 1.2-fold molar excess of pVIc prior to the crystallization trials. Diffraction-quality crystals were obtained at 18 degrees C by the vapor-diffusion method with 5.6 mg/ml AVP-pVIc in 1.4 M sodium acetate and 0.1 M Hepes, pH 7.5. Diffraction data (99% complete to 2.6 A resolution with Rmerge of 0.077) were collected from native crystals at room temperature at beamline X12-C at the National Synchrotron Light Source. The crystals belong to space group P6(1) with unit cell dimensions a = b = 114.2 A, c = 50.1 A; alpha = beta = 90 degrees, gamma = 120 degrees. The unit cell dimensions and likely mass of the molecular species in the crystals were consistent with there being one 25,000-Da complex (1:1) per asymmetric unit. Additionally, one heavy-atom derivative, obtained by the soaking of preformed crystals, was isomorphous to the native crystal. Diffraction data obtained on these crystals were 95% complete to 3.0 A resolution with an Rmerge of 0.076. Difference-Patterson analysis indicates three heavy atom sites in the derivative asymmetric unit.