Thrombotic thrombocytopenic purpura (TTP) is an uncommon disorder characterized by thrombocytopenia, schistocytic haemolytic anaemia, fever, neurologic lesions, and renal failure. A platelet aggregating factor has been postulated to be responsible for this disorder, but its precise identity remains debated. Two different groups of investigators have provided evidence that the platelet aggregating factor is a cysteine proteinase. We have suggested that it was calpain, whereas others have suggested that it was calpain, whereas others have suggested that it was cathepsin L. To help resolve this issue, we have studied the platelet activating activity found in the acute serum samples from 10 different TTP patients as well as purified calpain and cathepsin L. The TTP activity was measured functionally (platelet serotonin release assay and casein lysis assay) and antigenically (immunodepletion using anti-calpain and anti-cathepsin L antibodies and antigenic analysis using SDS PAGE). The TTP serum paralleled the activity of the purified calpain and had optimal pH activity of 7.5. The purified cathepsin L activity had optimal activity at low pH (5.5) and activity was no longer measurable at pH 7.5. Similarly, a specific cathepsin L inhibitor (Z-Phe-Phe-CHN2) had no effect on the activity of the TTP samples nor on purified calpain, but it did abolish the activity activity of purified cathepsin L. The platelet activating of the TTP samples was detectable in the microparticle pellet following ultracentrifugation of TTP serum, and could be immunodepleted using antibodies to calpain but not to cathepsin L. These studies indicate that the microparticle-associated platelet activating factor in TTP corresponds to calpain.