The antigen processing‐defective mutant cell line RMA‐S expresses at the cell surface major histocompatibility complex (MHC) class I molecules devoid of peptide that can be efficiently loaded with exogenous immunogenic peptides. We now report that viral peptide‐loaded RMA‐S cells, unlike parental RMA cells, can induce primary cytotoxic T lymphocyte (CTL) responses in vitro, in a T helper cell‐independent fashion. This was shown for an H‐2Kb‐binding peptide of Sendai virus nucleoprotein and an H‐2Db‐binding peptide of adenovirus type 5 E1A protein with responding spleen cells of C57BL/6 mice, the strain of origin of RMA and RMA‐S cells. Primary Sendai peptide‐induced CTL lyse both peptide‐loaded and virus‐infected cells. Pre‐culture of RMA‐S cells at low temperature (22°–26°C), which increases the amount of empty MHC class I molecules at the cell surface, decreases the peptide concentrations required for the induction of primary CTL responses. Primary peptide‐specific CTL responses induced by peptide‐loaded RMA‐S cells are CD4+ cell‐ and MHC class II+ cell‐independent. CTL response induction is blocked by the presence of anti‐CD8 monoclonal antibody during culture. Direct peptide binding studies confirm the efficient loading of empty MHC molecules on RMA‐S cells with peptide and show 2.5‐fold more peptide bound per RMA‐S cell compared to RMA cells. An additional factor explaining the difference in primary response induction between RMA and RMA‐S cells is related to the CD8 dependence of these responses. MHC class I molecules occupied with irrelevant peptides (a majority present on RMA, largely absent on RMA‐S) may interfere in the interaction of the CD8 molecule with relevant MHC/peptide complexes. The results delineate a novel strategy of peptide based in vitro immunization to elicit CD8+ cytotoxic T cell responses.