The usage of T-cell-receptor (TCR) alpha beta variable (V) regions among tumor infiltrating lymphocytes (TILs) in primary human malignant melanomas was characterized using a method based on the polymerase chain reaction (PCR). A panel of 57 different variable-region primers specific for the TCR V alpha I-29 and V beta I-28 was designed, and a semi-quantitative PCR method applicable to formalin-fixed, paraffin-embedded tissues was developed. This semi-quantitative method was demonstrated to be reproducible and to be useful for the assessment of V alpha- and V beta-gene-family usage in formalin-fixed, paraffin-embedded tissue samples. A total of 9 different histopathologically characterized primary tumors were analyzed in this study. The TILs in these tumors were found to preferentially express certain TCR V alpha and V beta regions. The differential usage of certain V alpha regions was very pronounced as illustrated by V alpha 4, which was highly expressed in 3/8 tumors, and V alpha 22, which was highly expressed in 4/8 tumors. For comparison, specific highly expressed V alpha regions in control samples of peripheral-blood lymphocytes rarely exceeded 10%. The most highly expressed V beta region was V beta 8, which was highly expressed in 2/8 tumors. For the highly expressed V alpha 4 and V alpha 22 and V beta 8 regions, the high levels may be explained by the in situ clonal or oligoclonal expansion of TIL. In one specific case, the high expression of V beta 8 was due to expansion of a single clone of TILs, as evidenced by a fully readable sequence of the CDR3 (V-D-J) region, determined by direct sequencing of the PCR product corresponding to V beta 8. In contrast, sequence analysis of V alpha 22, which was expressed in the same tumor sample at similar levels, demonstrated the simultaneous presence of 3 different CDR3 (V-J) sequences derived from V alpha 22 transcripts of exactly the same length. The observed preferential use of TCR V alpha and V beta regions suggests the in situ clonal expansion of specific populations of T-cells, possibly reactive with melanoma-associated peptides presented by HLA molecules. The preferential use of TCR V alpha and V beta regions may imply the involvement of a limited number of shared melanoma-associated peptides.