In this paper we examined the effect of anti-TNP antibody on guinea pig T cell proliferation in response to TNP-modified macrophages in vitro. The addition of anti-TNP to TNP-modified macrophages immediately after conjugation inhibited their ability to stimulate TNP-specific T cell proliferation. This inhibition appeared to be specific for the TNP response since anti-TNP had no effect on the ability of TNP-modified macrophages pulsed with either PPD or TNP-Ova to stimulate efficient PPD or Ova T cell responses. On the other hand, anti-TNP had no effect on the TNP-specific response to TNP-modified macrophages that had been cultured overnight before addition to primed T cells or to macrophages which had been pulsed with TNP-Ova. We also demonstrated that the same TNP-specific T cell subpopulation responds to both freshly TNP-modified macrophages and overnight cultured TNP-modified macrophages. These results suggest that the relevant TNP-determinants recognized by T cells are not exposed on the macrophage surface and raise the possibility that macrophages must process membrane-conjugated TNP to create the immunogen recognized by T cells.