Human T-cell leukemia virus type 2 (HTLV-2) was first isolated from leukemia patients, but has been found to be endemic among asymptomatic groups worldwide, including certain American Indian tribes (1). The virus infection is associated with a low incidence of disease among infected subjects, but has been found in patients with neurologic disorders and contributes to bacterial sepsis in AIDS patients (2,3). Polymerase chain reaction (PCR) and virus isolation techniques revealed that a high percentage of HTLV seroreactivity among intravenous drug users and blood donors in the United States is caused by HTLV-2 (4). Among serologic methods, enzyme-linked immunosorbent assays (ELISA) using whole virus preparations or in combination with recombinant and synthetic peptides are used as a primary screen for the infection. Antigen-capture systems have increased the sensitivity and accuracy in verification of HTLV-2 culture systems. The verification of HTLV-2 infection and detection of new strains of related viruses has been enhanced by employing virus-isolation methods using primary lymphocytes. Lymphocyte culture methods have also been used to test transformation properties of the virus and create stably expressing cell lines. This chapter briefly summarizes the biology of HTLV-2 infection and disease and details methods to isolate and verify the virus in lymphocyte cultures.