Chronic immune thrombocytopenic purpura (ITP) is an autoimmune hematologic disorder characterized by the presence of antiplatelet antibodies which lead to accelerated platelet clearance by the reticuloendothelial system. Although the platelet antibodies are produced by autoreactive B lymphocytes, there is also evidence to implicate T lymphocytes in the pathogenesis of this autoimmune disorder. We identified two unrelated males with chronic ITP who had highly elevated numbers of circulating Vβ8+ peripheral blood T lymphocytes documented on multiple occasions. Patient 1 had 8-12% Vβ8+ T lymphocytes, predominantly CD4+ (normal 2-4%), while patient 2 had 15-35% Vβ8+ T lymphocytes, almost exclusively CD8+. Total RNA was isolated from peripheral blood lymphocytes, and used for RT-PCR analysis and nucleotide sequencing of Vβ8-(D)-J-C gene rearrangements: For patient 1, 8 of 19 transcripts had an identical in-frame Vβ8.2-Dβ1.1-Jβ1.2-Cβ1 gene rearrangement, indicating a clonal expansion. Similarly, patient 2 had 8 of 23 mRNA transcripts with a Vβ8.3-Dβ1.1-Jβ1.2-Cβ1 in-frame rearrangement, and 7 of 23 with an in-frame Vβ8.2-Jβ1.1-Cβ1 rearrangement. Other Vβ8-(D)-J-C mRNA transcripts from these patients, as well as those from controls, demonstrated unique rearrangements using random combinations of N, Dβ, and Jβ segments. Patient 1 underwent splenectomy with resolution of his thrombocytopenia, along with normalization of the% peripheral blood Vβ8+ T lymphocytes. RT-PCR analysis of peripheral blood lymphocyte cytokine expression demonstrated elevated amounts of TNF-α, IL-2, andγ-IFN mRNA which decreased after splenectomy. Histologic staining of the splenic tissue revealed Vβ8+ T lymphocytes within the germinal centers, and RT-PCR identified the clonal Vβ8 sequence within the spleen. These results provide strong evidence that clonal T lymphocyte populations are present in chronic ITP, and are important in the pathogenesis of this autoimmune disorder.