An alternative pathway for class I-restricted antigen presentation has been suggested on the basis of peptides bound to HLA-A2 molecules in cells lacking the transporter for antigen presentation (TAP). Most of these peptides were derived from signal sequences for translocation into the endoplasmic reticulum (ER). However, it is not known whether these peptides can be presented to T cells. The hydrophobic nature of an HLA-A2-restricted T cell epitope (M1 58-66) was exploited to test whether it could be presented to T cells when derived from a signal sequence. Replacing the signal sequence of the influenza virus hemagglutinin molecule H3 with an artificial sequence containing that HLA-A2-restricted T cell epitope resulted in efficient translocation of H3 molecules into the ER and transport to the cell surface. This signal sequence-derived epitope was presented to HLA-A2-restricted T cells. Involvement of cytosolic processing for this presentation is very unlikely, because (a) presentation occurred in cells lacking TAP; (b) expression of H3 molecules with the artificial signal sequence did not produce a detectable cytosolic form of H3; and (c) presentation of the same epitope expressed in cytosolic forms of antigen required TAP. Thus, a peptide derived from a signal sequence cleaved in the ER can provide an epitope for HLA-A2-restricted T cell recognition.