We have developed a solid-phase competitive radioimmunoassay for human osteonectin, using a monoclonal antibody to bovine osteonectin. The assay is both specific and sensitive, being capable of measuring as little as 10 ng of osteonectin. Osteonectin measurements in parallel serum and plasma samples obtained from healthy individuals showed the plasma level to be 0.9 microgram/ml, while that of serum was 3 times higher, 2.6 micrograms/ml. Radioimmunoassay of blood cells indicated that platelets contain osteonectin at 1.9 micrograms per 2 X 10(8) cells. Further, the protein is released after thrombin stimulation of these cells. Immunoblot analyses of washed pelleted human platelets resulted in the identification of a single immunoreactive species. The molecular weight of this immunoreactive species was identical to that obtained for purified bovine bone osteonectin. The isolation procedure developed for bovine bone osteonectin was applied to human platelets and bone. The individual steps of the isolation procedure yielded identical profiles of immunoreactive material for bone and platelet extracts. Results of reverse-phase high-pressure liquid chromatography of bone- and platelet-derived osteonectin are consistent with the conclusion that the two sources yield the identical protein.