The conditions required for sensitizing naive T cells to nominal antigen are poorly understood. In this report we describe an in vitro system for generating antigen-specific CD4+ T cells from previously unprimed individuals. Freshly isolated CD4+ T cells were cultured with keyhole limpet hemocyanin (KLH), sperm whale myoglobin (SWM), or human immunodeficiency virus (HIV) gp160, antigens to which most persons have not been sensitized, in the presence of either dendritic cells (DC) or macrophages (M phi). In short-term (< 8 days) cultures, CD4+ T cells or their CD4+, CD45RA (naive) subpopulation mounted significant proliferative responses to KLH, SWM, and HIV gp160, but only if the antigens were presented by DC. In contrast, CD4+, CD45RO (memory) T cells responded poorly to these antigens, although they responded vigorously to tetanus toxoid, a recall antigen, presented by either DC or M phi. KLH- and SWM-specific CD4+ T cell lines were established from the starting population that had been sensitized in vitro, following repeated stimulation with antigen and M phi in medium supplemented with interleukin-2 and interleukin-4. Despite the continued presence of these cytokines during T cell expansion, the expanded lines retained their ability to respond to the priming antigen in the absence of exogenous cytokines. When the CD45RA and CD45RO subpopulations were sensitized and expanded separately, the CD45RA cells alone gave rise to antigen-specific T cell lines, while the CD45RO cells proliferated nonspecifically. These results demonstrate that human naive CD4+ T cells can be sensitized in vitro to nominal antigens presented by DC and that the sensitized cells can be expanded into long-term lines that retain their antigen specificity.