The ability of minigene-encoded viral peptide epitopes to be presented by class I molecules in the absence of MHC-encoded transporters has been evaluated in mutant T2 cells. These cells have a large deletion in the class II MHC region that includes the known transporter protein for antigenic peptides and proteasome genes and they are defective in presenting viral epitopes to CTL. T2 cells that express minigenes encoding the influenza virus matrix peptide 58-66 (GILGFVFTL) and two HTLV 1 Tax peptides 11-19 (LLFGYPVYV) and 12-19 were lysed by HLA-A2-restricted peptide-specific CTL. Minigene expression of a HLA-A2-restricted HIV reverse transcriptase peptide 476-484 (ILKEPVHGV) with three charged residues sensitized T2 cells poorly for lysis by HIV-specific CTL unless the peptide was preceded by an endoplasmic reticulum translocation signal sequence. Expression of an influenza virus nucleoprotein peptide 383-391 (SRYWAIRTR) with three charged arginine residues did sensitize HLA-B27+ T2 cells for lysis by peptide-specific CTL. These and other results with endogenously expressed peptide analogs in which hydrophobic and charged amino acids were interchanged demonstrate that antigenic peptides can be translocated from the cytoplasm into the class I Ag presentation pathway independent of MHC-encoded transporters; and that peptide hydrophobicity appears not to be a major determinant in selecting peptides for this alternate pathway.