The crystal structures of porcine pancreatic alpha-amylase isozyme II (PPA II) in its free form and complexed with the trestatin A derived pseudo-octasaccharide V-1532 have been determined using Patterson search techniques at resolutions of 2.3 and 2.2 angstroms, respectively. Seven rings of the competitive inhibitor V-1532 could be detected in the active site region as well as two maltose units in secondary binding sites on the surface. V-1532 occupies the five central sugar binding subsites similar to the PPA/acarbose structure. A sixth ring exists at the reducing end, connecting two symmetry related PPA molecules. The seventh moiety, a 6-hydroxymethylconduritol ring, is located at the non-reducing end. The electron density for this ring is relatively weak, indicating considerable disorder. This study shows that PPA is able to accommodate more than five rings in the active site region, but that additional rings would increase the binding affinity only slightly, which is in accordance with kinetic experiments. A comparison of the structures of free PPA, PPA/V-1532 and PPA/Tendamistat shows the characteristic conformational changes that accompany inhibitor binding and distinguish pseudo-oligosaccharide inhibitors from proteinaceous inhibitors. Although both classes of inhibitors block the sugar binding subsites in the active site region, the extreme specificity and binding affinity of the proteinaceous inhibitors is probably due to an intricate interaction pattern involving areas further away from the catalytic center.