Recently 10 T-cell lines were established from patients with adult T-cell leukemia (ATL). During establishment of these cell lines, it was found that when T-cell lines expressing the ATL-associated retroviral antigen were cocultivated with 8C cat cells, multinucleated syncytia were formed. Retroviral antigen-negative T-cell lines did not induce syncytia. Peripheral blood lymphocytes obtained from ATL patients did not express the retroviral antigen before cultivation in vitro but became positive for the retroviral antigen after cultivation for a short period; these retroviral antigen-positive lymphocytes, but not retroviral antigen-negative lymphocytes, induced syncytia upon cocultivation with 8C cells. Peripheral blood lymphocytes isolated from patients with chronic lymphocytic leukemia of T-cell origin or Sézary syndrome or from normal adults and lymph node cells from a patient with immunoblastic lymphadenopathy-like T-cell lymphoma did not express the retroviral antigen even after cultivation in vitro and did not induce syncytia upon cocultivation with 8C cells. Thus, there was complete agreement between the presence of the retroviral antigen in established T-cell lines or freshly isolated peripheral blood lymphocytes and their ability to induce syncytia. Syncytia formation was enhanced 5- to 20-fold by the presence of Polybrene and inhibited by addition of plasma of ATL patients to the cocultures. Syncytia were detected within 4 hr on cocultivation of 8C cells with the retroviral antigen-positive T-cells, indicating that most syncytia were formed by early polykaryocytosis. After cocultivation, a clone of 8C cells that harbored the ATL virus genome and had syncytia-inducing activity was isolated. These findings indicate that the retrovirus associated with ATL has syncytia-inducing activity. Syncytia induction assay using 8C cells will be useful for detection and characterization of human retrovirus associated with T-cell malignancies.