Platelet factor 4 (PF4), the platelet antiheparin protein, was isolated from both the supernatant and the cells of recently outdated platelet concentrates. Following purification by affinity chromatography, a competitive binding radioimmunoassay was developed to detect this protein in human plasma. The normal range was determined to be 9.4 +/- 4.7 ng/ml (mean +/- SD for 52 healthy adults). In order to determine whether individuals with transient ischemic attack (TIA) or stroke had measurable increments of PF4 in their plasma, radioimmunoassay studies were performed on 11 patients with well-documented TIA, 10 patients with well-documented stroke and on 16 age-matched controls hospitalized on a neurology service with disorders unrelated to arterial thrombosis. The 16 hospitalized controls had PF4 levels of 10.3 +/- 9.1 ng/ml, a value not significantly different from the 52 normals (P greater than 0.50). Patients with TIA had PF4 levels of 24.6 +/- 12.1 ng/ml, a value significantly higher than both the 52 normals (P less than 0.001) and the 16 hospitalized control patients (P less than 0.005). Patients with stroke had PF4 levels of 35.4 +/- 29.2 ng/ml, a value significantly higher than both the 42 normals (P less than 0.001) and the 16 hospitalized control patients (P less than 0.005). Outdated platelet concentrates facilitate the development of a reproducible radioimmunoassay for PF4. The elevation of this platelet-derived protein in the plasma of patients with stroke and TIA provides evidence for recent or ongoing platelet activation in the cerebral vascular disease population.