In a recent clinical trial, HLA-A2+ melanoma patients were vaccinated with a peptide derived from the melanoma Ag gp100, which had been modified at the second position (g9-209 2M) to enhance MHC binding affinity. Vaccination led to a significant increase in lymphocyte precursors in 10 of 11 patients but did not result in objective cancer responses. We observed that some postvaccination PBMC cultures were less reactive with tumor cells than they were with g9-209 peptide-pulsed T2 cells. In contrast, g9-209-reactive tumor-infiltrating lymphocyte cultures generally reacted equally with tumor cells and g9-209 peptide-pulsed T2 cells. To investigate this difference in T cell reactivity, T cell cloids derived from the PBMC of three patients vaccinated with g9-209 2M were compared with T cell cloids isolated from g9-209-reactive TIL cultures. All of the T cell cloids obtained from TIL reacted with HLA-A2+, gp100+ melanoma cell lines as well as with g9-209 and g9-209 2M peptide-pulsed targets. In contrast, only 3 of 20 PBMC-derived T cell cloids reacted with melanoma cell lines in addition to g9-209 and to g9-209 2M peptide-pulsed targets. Twelve of twenty PBMC-derived cloids reacted with g9-209 and g9-209 2M peptide-pulsed targets but not with melanoma cell lines. And 5 of 20 PBMC-derived cloids recognized only the g9-209 2M-modified peptide-pulsed targets. These results suggest that immunizing patients with the modified peptide affected the T cell repertoire by expanding an array of T cells with different fine specificities, only some of which recognized melanoma cells.