Factor V activity in suspensions of human platelets washed by albumin density gradient separation increased in response to stimulation by both collagen and adenosine diphosphate (ADP). The appearance of factor V activity extracellularly had the characteristics of platelet secretion and was partially inhibited by aspirin and by the antimetabolites 2-deoxyglucose and antimycin A. Some increase in factor V activity was also observed in platelet suspensions during the initial response to ADP; this activity was not detected extracellularly, but remained associated with the platelets. Patients with storage pool deficiency (SPD) whose platelets are deficient only in dense granule substances released normal amounts of factor V activity, whereas decreased amounts were released in a patient whose platelets have both dense and alpha granule deficiencies. These findings suggest that a portion of platelet factor V is associated with, and released from, alpha granules.