We investigated whether exposure of naive and in vitro pre-activated T cells to CD4 monoclonal antibodies (mAb) could induce specific hyporesponsiveness to a subsequent challenge in the absence of CD4 mAb. Unfractionated peripheral blood mononuclear cells were cultured with mitomycin-treated B cell lines as stimulator cells, in the presence or absence of CD4 mAb, then challenged with the same or unrelated stimulator cells. The kinetics of [3H] thymidine incorporation, blast transformation and CD25 expression were determined. Cells activated in primary or secondary culture in the presence of CD4 mAb demonstrated a markedly decreased response to subsequent challenge in the absence of antibody. This effect was reproduced with three different CD4 mAb of the IgG1 and IgG2a subclasses, which recognize two distinct epitopes of the CD4 molecule. Addition of recombinant interleukin-2 (rIL-2) during exposure to CD4 mAb failed to prevent the induction of specific hyporesponsiveness. Similarly, exogenous rIL-2, added together with stimulating cells, failed to restore the specific proliferative response, indicating that the mechanisms were different from those of classical anergy. The hyporesponsiveness was clonally restricted since CD4 mAb-pretreated cells developed a normal primary response to third-party stimulator cells. No increase in the percentage of apoptotic cells was observed in hyporesponsive cell populations, but selective clonal deletion cannot be excluded. The data demonstrate a delayed effect of CD4 ligation on T cell responses to a subsequent challenge.