The important immunological activities of Thymosin alpha 1 (T alpha 1), a peptide derived from the thymus, led to its use in combination therapies in cancer patients. Prothymosin alpha (ProT alpha) is a highly acidic polypeptide, first isolated as the putative precursor of T alpha 1. However ProT alpha is now known to be more immunoreactive than T alpha 1 in certain in vivo and in vitro assays. Recent results indicate that ProT alpha may be useful to design future therapeutic interventions in cancer patients if the mechanisms underlying these effects are puzzled out. With this in mind, we radiolabeled ProT alpha to obtain a high specific activity and a high biological activity for 125I-ProT alpha. Moreover, we also obtained autoantibodies exhibiting high titers and an unique specificity for anti-ProT alpha and anti-T alpha 1. With both tools we studied the presence of binding sites for ProT alpha on the surface of lymphoblast cells. We conclude that ProT alpha binds through the non-T alpha 1 sequence.