The identity of allogeneic peptide/major histocompatibility complex (MHC) complexes that elicit vigorous T cell responses has remained an interesting problem for both practical and theoretical reasons. Although a few abundant MHC class I-bound peptides have been purified and sequenced, identifying the unique T cell-stimulating peptides from among the thousands of existing peptides is still a very difficult undertaking. In this report, we identified the antigenic peptide that is recognized by an alloreactive bm1 anti-B6 T cell clone using a novel genetic strategy that is based upon measurement of T cell receptor occupancy in single T cells. Using lacZ-inducible T cells as a probe, we screened a splenic cDNA library in transiently transfected antigen-presenting cells (APCs) and isolated a cDNA clone that allowed expression of the appropriate peptide/Kb MHC complex in APC. The antigenic octapeptide (SVVEFSSL) exactly matched the consensus Kb MHC motif, but was surprisingly encoded by a non-ATG defined translation reading frame. Furthermore, the abundance of the naturally processed analog in untransfected cells was estimated to be <10 copies per cell. These results illustrate a novel strategy for identifying T cell-stimulating antigens in general and directly show that alloreactive T cells can respond to rather rare peptide/MHC complexes. These results also suggest that the total pool of processed peptides expressed on the APC surface may include those generated by cryptic translation of normally expressed transcripts.