Human T-cell lymphotrophic virus type 2 (HTLV-2), a common infection of intravenous drug users and subpopulations of Native Americans, is uncommon in the general population. In contrast with the closely related HTLV-1, which is associated with both leukemia and neurologic disorders, HTLV-2 lacks a strong etiologic association with disease. HTLV-2 does shares many properties with HTLV-1, including in vitro lymphocyte transformation capability. To better assess the ability of HTLV-2 to transform lymphocytes, a limiting dilution assay was used to generate clonal, transformed lymphocyte lines. As with HTLV-1, the transformation efficiency of HTLV-2 producer cells was proportionately related to the number of lethally irradiated input cells and was comparable to HTLV-1-mediated transformation efficiency. HTLV-2-infected cells were reproducibly isolated and had markedly increased growth potential compared to uninfected cells; HTLV-2 transformants required the continued presence of exogenous interleukin 2 for growth for several months and were maintained for over 2 years in culture. All HTLV-2-transformed populations were CD2 and/or CD3 positive and B1 negative and were either CD4+ or CD8+ populations or a mixture of CD4+ and CD8+ lymphocytes. Clonality of the HTLV-2 transformants was confirmed by Southern blot analysis of T-cell receptor beta chain rearrangement. Southern blot analysis revealed a range of integrated full-length genomes from one to multiple. In situ hybridization analysis of HTLV-2 integration revealed no obvious chromosomal integration pattern.