An enzyme-linked immunosorbent assay was developed to quantitate the platelet secretory glycoprotein, thrombospondin (TSP), in plasma. Specificity of the assay was provided by using an anti-TSP monoclonal antibody (McAb) to coat 96 well polystyrene assay plates. The effective range of the standard curve for the assay was between 2.5 ng/mL and 500 ng/mL. Interassay and intraassay variations were 6.9% and 7.5%, respectively. To avoid spontaneous release of TSP from platelets after the blood was drawn, and to obtain a valid measurement of plasma TSP concentration, it was essential to place and maintain blood samples on ice immediately after blood was drawn and to separate plasma from cellular constituents within two hours. The mean plasma TSP concentration of 24 healthy adults was 64.3 +/- 18.0 ng/mL (mean +/- SD). The TSP concentration of pooled normal human serum (n = 6) was 15.9 micrograms/mL. Interference occurred when undiluted plasma or serum samples were assayed. This interfering effect could be eliminated completely by a tenfold dilution of plasma or serum samples before the assay. The assay also was applied successfully to quantitate TSP release from platelets after induction of aggregation by different platelet-aggregating agents.