Characterization of tumor-associated antigens (TAAs) recognized by CTLs makes the consideration of therapeutic strategies based on peptide stimulation of peripheral blood lymphocytes (PBLs) feasible. Several such approaches are adoptive transfer of peptide-stimulated PBLs, ex vivo peptide stimulation of dendritic cells, and direct vaccination with TAA-derived peptides. A critical component of any of these peptide-based strategies is the requirement that the patient's PBLs are able to react productively against the presented TAA. The purpose of this study, through the study of T-cell receptor (TCR) usage, was to evaluate the T-cell response in matched MART-1(27-35) peptide-stimulated PBLs and tumor-infiltrating lymphocytes (TILs). MART-1(27-35)-reactive PBL and TIL cultures were generated from three patients by in vitro stimulation with an immunodominant peptide of MART-1 (MART-1(27-35)). All cultures had a human leukocyte antigen A2-restricted, MART-1(27-35)-specific CTL response. The TCR usage of each was assessed by the DNA sequence analysis of 50 TCR beta clones obtained by rapid amplification of cDNA ends per culture. TCR analysis suggests a TCR repertoire that differed from patient to patient (8-16 subfamilies were used) and a predominant usage of a different variable beta chain (BV) by each of these MART-reactive T cells. These predominant BV rearrangements were derived from multiple clonotypes because different variable, diversity, and junctional regions were observed. However, a similar pattern of expansion was present for both PBLs and TILs; the relative usage of each prevailing BV was more marked in TILs (36, 50, and 78% of TILs versus 26, 20, and 24% of PBLs, respectively), a broader TCR repertoire was used by PBLs (P > 0.05), and similar TCR subfamily usage was noted when TIL and PBL cultures from the same patient were compared (8 of 11, 7 of 9, and 7 of 8 for patients 1, 2, and 3, respectively). Furthermore, the exact same clonotypes derived from predominant TCR subfamilies in the PBLs and TILs were present in each patient, suggesting peptide-stimulated expansion in both biological compartments. These studies suggest that there will not be a limited and predictable TCR subfamily response to a specific TAA, although reproducible patterns of PBL and TIL expansion are present from patient to patient. Additionally, identical T-cell clonotypes having the same potential for antigen-driven expansion were present in a patient's PBLs and TILs. As such, our data support the conceptualization of approaches using adoptive transfer or vaccination based on TAA-derived peptide stimulation of PBLs.