An Immunoradiometric assay for thymosin alpha 1(1-28) was developed using a monoclonal and a polyclonal antiserum which were raised against synthetic thymosin alpha 1(1-28) and thymosin alpha 1(16-28), respectively. A monoclonal antibody, specific for the (acetylated ser1) 1-5 sequence of thymosin alpha 1, was immobilized on polystyrene beads for the solid phase, and a polyclonal antiserum specific for the 16-28 sequence was employed. This method relies on the formation of an immune complex consisting of a 125I-labelled anti-rabbit IgG goat antibody, polyclonal antiserum, thymosin alpha 1, and the solid phase monoclonal antibody. Radioactivity on the solid phase is directly proportional to the amount of thymosin alpha 1 present in the specimen. The minimal detection limit of this assay system was approximately 1.9 pg/ml. The mean values of thymosin alpha 1 in plasma of healthy subject, ranging in age from 0 to 3 years was approximately five fold higher than that of higher ages. HPLC analysis of plasma of a healthy subjects revealed a single immunoreactive form which eluted with the same retention time as that of synthetic thymosin alpha 1. This assay will be extremely useful for the measurement of thymosin alpha 1 in biologic fluids and tissues.