CTL recognize peptide forms of processed, foreign antigens in association with class I molecules encoded by the MHC and are usually directed against endogenously synthesized "cellular antigens," such as those expressed by virus-infected cells. In vitro studies have shown that small exogenous peptides can directly associate with class I molecules on the cell surface and mimic the target complex derived by intracellular processing and presentation. We have recently generated OVA-specific, H-2Kb-restricted CTL by immunizing C57BL/6 mice with a syngeneic tumor line transfected with the OVA cDNA. The CTL recognize the OVA transfectant E.G7-OVA and the synthetic peptide OVA258-276, but fail to recognize the native protein. We reasoned that given the potential for direct peptide/class I association observed in vitro, OVA258-276 may induce CTL after in vivo priming. However, we found that this is not the case. OVA258-276 and peptides of increasing lengths up to OVA242-276 and OVA242-285, which are all able to form the target complex in vitro, are inefficient at priming E.G7-OVA-specific CTL responses after intravenous injection. This is also true for both native and denatured OVA. In contrast to these results the synthetic peptide OVA229-276 corresponding to a peptide in a partial tryptic digestion of OVA can efficiently prime C57BL/6 mice in vivo after intravenous injection. This peptide elicits CTL that appear identical to those derived from animals immunized with syngeneic cells producing OVA endogenously. These results are discussed in terms of separate class I and class II antigen presentation pathways and the ability of only certain, exogenous antigens to enter the cytoplasmic, class I pathway.