Thymopoietin is a thymic hormone that induces differentiation of thymocytes from precursor cells which arise in hemopoietic tissues. This paper describes a sensitive in vitro assay for the induction of Thy 1.2 antigen on null lymphocytes from germ-free athymic (nu/nu) mice. The sensitivity and specificity of the bioassay were increased by adding high concentrations of ubiquitin (a nonspecific inducer) to the induction incubations. The bioassay was sufficiently sensitive to detect thymopoietin at less than 0.25 ng/ml. A dose-response relationship was shown between thymopoietin concentration and the percentage of cells induced to express Thy 1.2 antigen. When normal human plasma was assayed, induction was registered with activity corresponding to thymopoietin at greater than 1 ng/ml in plasma from infants or young adults. Activities in the thymopoietin range of 0.25 ng/ml were registered with plasma from healthy subjects over 50 years of age. Thymectomy was followed by loss of this inductive activity from the plasma. This bioassay permits clinical studies on T (thymus-derived) cell inducers released by the human thymus into the circulation.