The human T cell leukemia virus II (HTLV-II) is a type C retrovirus closely related to the human transforming retrovirus HTLV-I. In contrast to HTLV-I, the role of HTLV-II in human disease is controversial. However, HTLV-II infection has been documented in several cases of a clinically benign hairy cell leukemia and has also been suggested as a cofactor for HIV-1 disease progression. We report that an HTLV-II isolate (designated FLW) derived from a serum-positive white male can induce cell fusion and significant cytopathic effects in tissue culture. This HTLV-II isolate induced syncytium formation with human T and B cell lines, several human fibroblast cell lines, and, interestingly, HIV-1- and HIV-2-infected cell lines. To elucidate the role in the FLW envelope in this phenomenon, we have cloned the envelope glycoproteins gp46 and gp21 of this isolate. The envelope glycoproteins expressed in the absence of the rest of the viral genome were sufficient to drive syncytium formation in vitro, and preserved the cellular tropism for syncytium formation observed with the native retroviral isolate. Amino acid (aa) sequence analysis demonstrated 88% overall similarity with other HTLV-II envelope glycoproteins. Interestingly, only cells infected by HIV-I, but not parental H9 cells, form syncytia with FLW env-transfected cells as well as with HTLV-II/FLW-infected BJAB-WH cells. Furthermore, antibodies directed at the CD4 receptor failed to inhibit the induction of giant cell formation, implying that the FLW envelope protein was responsible for driving syncytium formation in this system. These observations may be important for the understanding of the processes involved in human retroviral-mediated syncytium formation and may suggest a mechanism whereby HTLV-II could influence the disease process in individuals dually infected with HIV-1 and HTLV-II.