Infection by human T-cell leukemia viruses (HTLV-I and HTLV-II) induces syncytium formation in certain cell types in vitro. Recombinant vaccinia viruses expressing the HTLV-II envelope (Env) gp61-II were used to infect human cells, and syncytium formation was observed, demonstrating that HTLV-II Env expression was sufficient to mediate syncytium formation in appropriate cells. This syncytium formation could be blocked by sera from HTLV-II-infected individuals. Infection with these recombinant vaccinia viruses caused fusion in mouse/human hybrid cells containing only human chromosome 17, whereas there was no obvious syncytium formation in mouse cells. This fusogenic phenotype of recombinant gp61-II is likely a property of the specific interaction between HTLV-II envelope protein and the HTLV cellular receptor, encoded by a gene that was previously indicated to be on human chromosome 17. On this basis, we developed a sensitive syncytium formation assay, using lacZ gene activation, for detecting the presence of the HTLV receptor(s). We used this fusion assay to test for HTLV receptor activity on a variety of cells. Our findings indicate that the HTLV receptor is widely distributed among species and cell types, including, to a limited extent, murine cells.