Human platelets contain 0.61 +/- 0.06 (S.E.) U of VIIIR:Ag per 10(9) platelets or about 25% of the circulating antigen in whole blood. Assay of subcellular fractions obtained by sucrose density-gradient centrifugation of disrupted platelets indicates that over 85% of the VIIIR:Ag is in the alpha granule fraction with other nonenzymatic proteins which can be released by treating platelets with collagen or thrombin. Seven percent or less is in the membrane fraction. After exposure of platelet suspensions to an optimal concentration of collagen, the supernatants contain, at most, 30% of the total amount of VIIIR:Ag in the platelets but a higher percentage of other releasable substances. After exposure to thrombin, the supernatants contain virtually no VIIIR:Ag. These low values are attributed to release of a limited amount of VIIIR:Ag by collagen or thrombin rather than to adsorption or destruction of antigen, for several reasons. (1) VIIIR:Ag can be recovered in the platelet residue as effectively as in control samples. (2) The antigen cannot be detected on the surface of collagen- or thrombin-stimulated platelets. (3) Relatively little released antigen is adsorbed by collagen or destroyed by thrombin. Release of VIIR:Ag by collagen is markedly reduced by aspirin, and release of the antigen as well as fibrinogen from platelets is much slower than release of 14C-serotonin and beta-TG.