Destabilase, endo-epsilon-(gamma-Glu)-Lys isopeptidase from the medicinal leech, inhibits arterial thrombus formation in rats. Inhibition of platelet aggregation was supposed to be one of the main mechanisms of this phenomenon. To elucidate this question highly purified destabilase preparations were used. Aggregation was monitored both by a turbidometric method and by a method based on real-time estimation of mean aggregate size. Spontaneous aggregation of human platelets was completely blocked by destabilase. At 5 microM ADP maximal inhibition was 63%. Aggregation induced by PAF (100 nM) and collagen (0.1 mg/ml) was inhibited in the presence of destabilase by 50 and 65%, respectively. This enzyme does not activate adenylate cyclase but inhibits it. We suggest that destabilase interacts with high-affinity binding sites on the platelet plasma membrane, thus providing an anti-aggregating effect. This idea coincides with the data that destabilase primary structure has high homology with some adhesive proteins.