An experimental system has been established to understand the poor interleukin-2 (Il-2) production by activated thymocytes. This model system is further characterized here and studies were done on the possible mechanism(s) involved. Thymocytes activated by Concanavalin-A (Con-A), or through the CD3 complex of the T-cell receptor (TCR), inhibit 95-99% of the Il-2 production by spleen cells, while thymocytes stimulated by rIl-2 or lipopolysaccharides (LPS) do not. The mechanism of inhibition is not due to production of soluble factors, consumption of available interleukin-1 (Il-1) or Il-2, but is dependent on cell-to-cell contact. Although cellular contact is needed, cytotoxicity is not involved. Prostaglandin production is not required for the generation or exertion of the inhibitory activity. Protein and DNA synthesis are necessary for exertion of the suppressive effect. We also demonstrate a genetic difference between different mouse strains in the ability to generate the inhibitory thymocytes. Activated Balb/c thymocytes inhibit spleen cells' Il-2 production in a non-MHC-restricted manner. Our studies demonstrate a regulatory capacity of activated thymocytes in vitro. This ability of the postnatal cells could be of relevance for understanding the latter events in T-cell education in the thymus and the role of Il-2 during this process.