We have shown that certain CD4+ T cell lines can function as suppressor cells in a cell culture system. In this context, the CD4+ T cells (AS-9) cloned from the peripheral blood lymphocytes (PBL) of a melanoma patient are capable of suppressing the induction of cytolytic response in autologous PBL in coculture. Here we show that a trypsin-sensitive cell-free culture supernatant factor from the AS-9 cells, AS-9 SF, interferes with IL-2 synthesis by T cells when they are stimulated. AS-9 SF also selectively blocks the expression of interleukin-2 receptor alpha (IL-2R alpha) on T cells during activation. Expression of transferrin receptors and the CD3 molecules is not down-regulated by this factor. The AS-9 SF consequently blocks proliferation of T cells when they are stimulated by lectin or activated through the T cell receptors. AS-9 SF suppresses the IL-2R alpha induction and the T cell proliferation at the induction phase only because it has no suppressive effect on preactivated T cells. Interleukin-2, IL-2R alpha, and beta messages are not down-regulated by the AS-9 SF and the suppressive effect of the AS-9 SF on IL-2R alpha expression and on T cell proliferation is not neutralized by the addition of exogenous recombinant IL-2. The factor does not appear to be IL-4, IL-10, or TGF-beta, three known cytokines possessing regulatory properties on T cell activation.