Interleukin 2 (IL-2) receptors which are expressed in T-lymphocytes after antigenic or mitogenic stimulation have been measured with either 3H- or 125J-labelled IL-2 or with receptor antibodies (e.g. anti-Tac). We have developed a new method which allows the determination of endogenously bound IL-2, or of the IL-2 binding capacity, respectively. The method utilizes incubation of target cells with human natural highly purified IL-2. Total bound IL-2 is determined by a conventional 3H-thymidine incorporation assay. An additional step, differential dissociation, eliminates low affinity bound IL-2 so that the remaining high affinity bound IL-2 can be determined. In PHA stimulated mononuclear cells we have found that high affinity binding sites appear after one day at high concentration and disappear gradually after more than ten days of culture. Low affinity binding sites appeared first on day 2, but showed then similar kinetics. IL-2 binding capacity closely correlated with the ability of the cells to respond to exogenous highly purified human IL-2. Our results demonstrate that in vitro in healthy blood donors the expression of IL-2 binding sites is necessary to render the cells responsive to IL-2 and to induce polyclonal expansion. Pharmacological doses of hydrocortisone abolishes both, IL-2 binding and IL-2 response.