Thymic stromal cells from newborn, 2 wk, 2 and 5 mo old BALB/c mice, which are adherent to plastic surface, were cultured in vitro for 3 weeks. The supernatants collected twice weekly were pooled and assessed for their ability to inhibit the proliferation of indicator cells stimulated with crude interleukin(IL)-2. The indicator cells were peanut agglutinin (PNA)+ and PNA- thymic T cells, splenic T cells, splenic T blast cells and cytolytic T lymphocytes line 2 (CTLL-2) cells, and they were all stimulated with crude IL-2 to proliferate. Supernatants from splenic and bone marrow stromal cells from 2 mo old BALB/c mice were cultured and were tested in a similar manner. The following results were obtained: (1) Supernatants of thymic stromal cells inhibited the proliferation of PNA+ and PNA- thymic T cells but not that of splenic T cells, splenic T blast cells and CTLL-2 cells; (2) The supernatants of splenic and bone marrow stromal cells had no inhibitory activity against PNA- thymic T cells stimulated with crude IL-2; (3) The gel elution patterns of nondialyzable inhibitory factors produced by thymic stromal cells of 2 and 5 mo old mice were polydispersed, in contrast to those produced by thymic stromal cells of newborn and 2 wk old mice; (4) Diethylaminoethyl (DEAE)-Sephadex ion exchange chromatography resolved the inhibitory factors produced by thymic stromal cells of 5 mo old mice into 3 peaks, and the major peak was estimated to be molecular weight (Mr) 68,000 based on Sephacryl S-300 gel filtration analysis; (5) This fraction was relatively heat stable; it inhibited the proliferation of crude IL-2 stimulated PNA- thymic T cells in apparently a nonstoichiometric manner; and bovine serum albulmin (BSA), which is present in the fraction, and interferon, which could be present in the fraction, had no inhibitory activity.