Bovine buffy coat cells infected with the bovine leukemia virus (BLV) induce syncytia formation in human diploid embryonic lung cells as well as in monolayer cell cultures of bovine, simian, ovine, bat, and caprine origin, but not in mouse fibroblast cells, normall rat kidney cells, or RSV-transformed rat cells. Syncytia were not observed in diploid embryonic lung cells inoculated with bovine buffy coat cells free of BLV. The syncytia-induction effect is associated with the synthesis of complete BLV by the buffy coat cells and is independent of whether these cells are viable, disrupted, normal, or malignant. Cell-free preparations of BLV and density gradient-purified virus also induce syncytia when added directly to diploid embryonic lung cells and to bovine, bat, and caprine monolayer cell cultures. Ether treatment, ultraviolet light irradiation, heating, freezing, and thawing destroy the syncytia-inducing activity of BLV. This activity is also neutralized when the virus is incubated with sera containing antibodies to BLV, but not when incubated with sera free of these antibodies or reference serum for the foamy-like bovine syncytial virus. Several other lines of evidence rule out the possibility that this virus or other bovine viruses are responsible for the syncytia-inducing phenomenon described here. BLV antigen was consistently detected by the immunofluorescent test in the syncytia-positive monolayer indicator cultures. However, syncytia formation was not necessarily associated with BLV production by the indicator cells. The ability to induce syncytia in monolayer cultures of nontransformed cells distinguishes BLV from all the known C-type luekemia viruses.