We have investigated the requirement for the presence of L3T4-positive T cells in the in vivo priming of Lyt-2-positive cytotoxic T-lymphocyte precursors. The antigens used were the male-specific antigen H-Y and autosomal minor histocompatibility antigens. In some experiments, responder mice were depleted of L3T4-positive cells by repeated intraperitoneal injections of the anti-L3T4 antibody, GK1.5. In other experiments, lymphoid cells from normal mice were fractionated in vitro by antibody-plus-complement treatment and the populations primed in irradiated adoptive hosts. In these antigen systems, depletion of L3T4-positive helper cells decreases the level of priming of cytotoxic T lymphocytes. With regeneration of the L3T4-positive subpopulation, the CTL response to antigen increases. To some extent, the reliance on L3T4-positive cells can be overcome by increasing the antigen dose. We conclude that in most physiological responses, L3T4-positive T cells enhance the cytotoxic T-cell response.