The conditions favouring effective specific cytotoxic T lymphocyte (CTL) priming have been exploited to set up a simple and reproducible method to induce a primary CTL response in vitro. We report that cultured monocytes, as well as activated T cells, pulsed with exogenous HLA-A2 binding immunogenic peptides, can induce primary peptide-specific CTL responses in vitro in a T_h-independent manner. Primary viral peptide-induced CTL were HLA-A2 restricted, and recognized both peptide-pulsed target cells and targets infected with recombinant vaccinia virus expressing viral endogenous antigens. In addition, both cultured monocytes and activated T cells primed peptide-specific CD8⁺ T cells depleted from the CD45RO⁺ memory cell fraction. The efficiency of CTL priming by monocytes was dependent upon the strong up-regulation of class I, adhesion and co-stimulatory molecules occurring spontaneously upon in vitro culture. The inability of unseparated peripheral blood mononuclear cells to mount a peptide-specific CTL response could be reverted by direct co-stimulation of responding CD8+ T cells by soluble B7.1 or a stimulatory anti-CD28 antibody, that allowed a specific response to take place. Although co-stimulation via the B7-CD28 interaction appeared sufficient to trigger CTL responses, It was not essential for CTL priming, since neither anti-B7.1 mAb nor soluble CTLA-4 inhibited induction of primary CTL response. This new method for induction of specific CD8⁺ T cell response in vitro may be exploited in adoptive immunotherapy in cancer or in HIV-infected patients.