A number of Ags recognized by tumor-reactive T cells have been characterized, including nonmutated gene products and a variety of epitopes shown to arise from either mutated or alternatively processed transcripts. Here, we report that the screening of a cDNA library with an HLA-B7-restricted renal cell carcinoma-reactive T cell clone derived from tumor-infiltrating lymphocytes (TILs) that were clonally amplified in vivo (as assessed by TCRBV complementarity determining region-3 length distribution analysis) resulted in the isolation of a nonamer encoded by an alternative open reading frame (ORF) (a +1 frameshift) of the intestinal carboxyl esterase gene. This peptide binds HLA-B*0702-presenting molecules as assessed in an immunofluorescence-based peptide binding assay using transfected T2 cells. Constitutive expression of this alternative ORF protein was observed in all transformed HLA-B7+ renal cell lines that were recognized in cytotoxicity assays by the TILs. The intestinal carboxyl esterase gene is transcribed in renal cell carcinoma tumors as well as in normal liver, intestinal, or renal tissues. Mutation of the natural ATG translation initiation site did not alter recognition, indicating that frameshifting (i.e., slippage of the ribosome forward) and recoding are not involved. In addition, a point mutation of the three AUG codons that may be used as alternative translation initiation sites in the +1 ORF did not abolish recognition, whereas mutation of an upstream ACG codon did, indicating that the latter codon initiates the translation of the alternative ORF. These results further extend the types of Ags that can be recognized by tumor-reactive TILs in situ (i.e., leading to clonal T cell expansion).