The antiphospholipid antibody syndrome is characterized by circulating antiphospholipid antibodies against cardiolipin (CL) and phosphatidylserine (PS) and clinically associated with a high risk of spontaneous thrombosis. Three monoclonal antibodies that differentiate between CL or PS were tested against resting and thrombin-activated platelets by flow cytometry. Each antibody reacted differently with CL and PS; 3SB9b reacted with PS, D11A4 reacted with CL, and BA3B5C4 reacted with both CL and PS. Activated platelets bound BA3B5C4 and 3SB9b, but not D11A4. The BA3B5C4-reactive epitope appeared earlier during activation than the epitope reactive with 3SB9b. These data suggest that antibodies against PS are reactive with activated platelets and that two immunoreactive forms of PS are sequentially expressed on platelets during activation.