Objectives: To characterize tumor-infiltrating lymphocytes (TILs) within lesions of cutaneous squamous cell carcinoma (SCC) and related disorders. Design: Case series with 1- and 2-color immunohistologic, molecular biological analysis of T-cell clonality and in vitro cytotoxicity assays. Setting: Academic medical center. Patients: Twenty-one patients, including 6 with actinic keratoses, 4 with SCC in situ, and 11 with invasive SCC. Results: CD8+ TILs were present within lesions of cutaneous SCC and AK. These cells constituted a variable minority of the total T-cell infiltrate, and many expressed a phenotype consistent with major histocompatibility complex-restricted cytotoxic T lymphocytes: CD3+, TIA1+, CD16-, CD56-, CD57-. They also expressed HLA-DR, suggesting their activation in vivo. Virtually all T cells were T-cell receptor (TCR)-β+ δ, indicating that they expressed the TCR-αβ protein heterodimer. Molecular biological analysis of TCR-γ gene rearrangements by the polymerase chain reaction and denaturing gradient gel electrophoresis technique indicated that the TILs were polyclonal. Functional studies suggested that TILs derived from SCC lesions were cytotoxic for autologous tumor cell targets. Conclusion: Tumor- infiltrating lymphocytes within cutaneous SCC lesions contain a subpopulation of polyclonal, major histocompatibility complex-restricted cytotoxic T lymphocytes expressing the TCR-αβ heterodimer.