With the goal of establishing an in vitro system of dual infection with human T cell lymphotropic viruses (HTLV) types I and II, rabbit lymphocytes were cocultured with a mixture of lethally irradiated HTLV-I-producing Ra-1 and HTLV-II-producing RII cell lines. This gave rise to a lymphoid cell line, RW-1, that was dually infected with HTLV-I and -II as detected by immunofluorescence staining, electron microscopy, and polymerase chain reaction using primers specific for the pol and env regions of each virus and by Southern blot hybridization. Two clonal cell lines derived from RW-1 were also coinfected with the viruses, indicating that dual infection had occurred at the single cell level. The coinfection could be readily propagated to fresh lymphocytes by coculture with RW-1. In contrast, attempts to superinfect HTLV-I-infected lymphoid cell lines with HTLV-II and vice versa were consistently unsuccessful, suggesting receptor interference between HTLV-I and -II.