Controversy exists as to whether anti-tumor immune responses are polyclonal in nature or, alternatively, result from the expansion of clonally restricted TCR specificities targeting the same melanoma-associated antigenic peptides. In order to address this question, we determined TCR usage in tumor-infiltrating lymphocytes of 14 melanoma specimens from three different HLA-typed melanoma patients (HH: nodular melanoma, lymph node, liver, lung, pancreas, brain, skin; GS: liver, lung; SP: lung, spleen, kidney, thyroid gland, skin - HH/SP: HLA-A24, B35, Cw4; GS: HLA-A3, B35, Cw4;) uniformly expressing the melanoma-specific antigens gp100 and Mart-1. Semiquantitative TCRV-specific PCR detected an overall predominance of only one TCRV gene segment family within all three melanoma patients. This is consistent with the hypothesis of a clonally expanded anti-melanoma immune response. However, subcloning and sequencing of the dominating TCRBV specificity revealed that only the primary tumor and a brain metastasis of patient HH harbor T-cells carrying a solitary TCRBVDJ sequence motif, whereas TIL of all other tumor specimens, although preferentially using the same combination of TCRBVJ families, express highly polymorphic CDR3 regions. Together with the identification of a gp100-specific TCRβ-chain motif within skin and lung metastases, our results favor the concept of a tumor-specific immune response against a rather limited number of melanoma-associated antigenic moieties in the patients investigated. We further postulate that this response does not result from expansion and dissemination of only few T-cell clones but rather from the multicentric activation of individual T-cells with different, yet related TCR specificities.