A broad array of antibodies directed against the variable (V) region of the T-cell receptor (TCR) beta (V beta) chain has become available in a directly conjugated multicolor format that permits assessment of 19 of 25 V beta families, covering 70% of the normal circulating T-cell repertoire. These antibodies were used to detect expanded T-cell populations in 43 peripheral blood samples submitted for suspected T-cell malignancy. Of 43 samples, 27 were diagnosed as follows: T-cell large granular lymphocyte leukemia, 14 samples; Sézary syndrome, 4 samples; T-cell prolymphocytic leukemia, 5 samples; or T-cell non-Hodgkin lymphoma or T-cell lymphoproliferative disorder not otherwise specified, 4 samples. The remaining 16 samples were determined to be nonneoplastic. All samples were diagnosed before assessment with anti-V beta flow cytometry. By using a cutoff of 1.6 times the upper limit of normal range (ULN) to define malignant restriction of V beta use, pathologic restriction of V beta use was found directly or indirectly in all 27 samples carrying a diagnosis of malignancy and directly in 2 of 16 samples without a diagnosis of malignancy. TCR gene rearrangement studies were used to confirm V beta flow cytometry results. By using a cutoff of 1.6 times the ULN for the detection of malignancy, the antibody panel had a diagnostic sensitivity of 89% for direct detection of pathologic V beta restriction and a specificity of 88%, making it useful for rapid diagnosis of T-cell leukemia.