A rat cDNA (rRam-l), which was cloned on the his that it enables Chinese hamster ovary (CHO) cells to be infected by amphotropic host range murine retroviruses, was recently found to encode a widely expressed Na+-phosphate symporter (M. P. Kavanaugh, D. G. Miller, W. Zhang, W. Law, S. L. Kozak, D. Kabat, and A. D. Miller, Proc. Natl. Acad. Sci. USA 91:7071-7075, 1994). CHO cells express the hamster homolog of Ram-1 but are resistant to amphotropic retroviruses. Although the amphotropic envelope glycoprotein gp70 bound weakly onto control CHO cells, CHO/rRam-1 cells had novel high-affinity binding sites, and the resulting strongly adsorbed gp70 was only slowly removed from cell surfaces, with a half-life of greater than 6 h. CHO/rRam-1 cells were also specifically and efficiently killed by exposure to amphotropic gp70 followed by antiserum to gp70 in the presence of complement. Infection with an appropriately pseudotyped form of amphotropic retrovirus 4070A did not perturb control CHO cells or inhibit their phosphate transport. In contrast, 4070A infection of CHO/rRam-1 cells caused major alterations including cell-cell fusions, a specific 40% down-modulation of the rRam-1 component of phosphate transport, and complete interference to super infection by amphotropic viruses. The 4070A virus-infected CHO/rRam-1 cells retained a substantial cell surface pool of rRam-1 that functioned as a phosphate transporter but not as a viral receptor. We conclude that amphotropic gp70 binds more strongly to rRam-1 than to the homologous hamster protein and that this stable attachment is necessary for infection, interference, membrane fusion, and pathogenesis.