Xenotropic and polytropic murine leukemia viruses (X-MLVs and P-MLVs) cross-interfere to various extents in non-mouse species and in wild Asian mice, suggesting that they might use a common receptor for infection. Consistent with this hypothesis, the susceptibility of some wild mice to X-MLVs has been mapped to the P-MLV receptor locus at the distal end of mouse chromosome 1. In this study, we report the isolation and characterization of a cDNA for the human X-MLV cell surface receptor (X-receptor) by using a human T lymphocyte cDNA library in a retroviral vector. The predicted X-receptor contains 696 amino acids with multiple hydrophobic potential membrane-spanning sequences and with weak homologies to the yeast proteins SYG1, of unknown function, and PHO81, which has been implicated in a system that regulates transport of inorganic phosphate. Expression of the X-receptor in Chinese hamster ovary cells, which are substantially resistant to P-MLVs and to X-MLVs, made them susceptible to both of these virus groups. The mouse homologue of the X-receptor was mapped by hybridization to the distal end of chromosome 1 at the same position as the P-MLV receptor gene Rmc1. These results strongly support the hypothesis that a common gene encodes the receptors for X-MLVs and P-MLVs, with the human X-receptor preferentially mediating X-MLV infections and the homologous protein of inbred mice mediating only P-MLV infections. We propose that X-MLVs and P-MLVs comprise a single family of retroviruses that have coevolved in response to diversification in X-receptor genes of the host.