Anti-lymphocyte and anti-thymocyte globulins (ATG) are currently used as immunosuppressive agents in organ transplantation. Their administration in vivo may induce not only lymphocyte depletion but also functional effects which were investigated in the present study. In vitro ATG inhibited T-cell proliferation induced by monocyte-dependent T-cell mitogens, like CD3 antibodies, phytohaemagglutinin (PHA) and concanavalin A (Con A), by monocyte-independent mitogens, like CD2 antibodies, or by protein kinase C activators (phorbol esters) associated with a calcium ionophore. The inhibitory effect of ATG was therefore not solely accounted for by a suppression of co-stimulatory signals delivered by monocytes, but rather implied a direct action on T cells. Addition of recombinant human interleukin-2 (rIL-2) did not overcome the inhibition. Suppression of T-cell proliferation by ATG was characterized by normal RNA synthesis and IL-2 secretion contrasting with markedly reduced expression of the CD25 protein [p55, the alpha-chain of interleukin-2 receptor (IL-2R)] both in cytoplasm and on T-cell membrane, as well as a decreased secretion of interferon-gamma (IFN-gamma). Northern blot analysis revealed increased levels of CD25 and IFN-gamma mRNA, suggesting a post-transcriptional inhibition of these molecules, whereas IL-2 mRNA levels were unchanged. These data demonstrate that inhibition of T-cell proliferation by ATG can be attributed primarily to a post-transcriptional defect of CD25 expression, implying a novel mechanism different from those described with other immunosuppressive agents. Blocking of T-cell proliferation in the late G1 phase of the cell cycle may contribute to the immunosuppressive activity of ATG in prophylactic treatment of allograft rejection.