A Mus dunni tail fibroblast (MDTF) cell line is highly resistant to infection by ecotropic Moloney murine leukemia virus (Mo-MLV). The cationic amino acid transporter type 1 (CAT1) paralogues of murine NIH 3T3 and MDTF cells (mCAT1 and dCAT1, respectively) contain two conserved N-linked glycosylation sites in the third extracellular loop (ECL3, the putative Mo-MLV binding site). Glycosylation of dCAT1 inhibits Mo-MLV infection, but that of mCAT1 does not. Compared with mCAT1, dCAT1 possesses an Ile-to-Val substitution at position 214 and a Gly insertion at position 236 in the ECL3. To determine the residues responsible for the loss of dCAT1 receptor function, mutants of mCAT1 were constructed. The mCAT1/insG receptor (with a Gly residue inserted at mCAT1 position 236) had greatly reduced Mo-MLV receptor function compared with mCAT1. Treatment of mCAT1/insG-expressing cells with tunicamycin, an N-linked glycosylation inhibitor, increased the transduction titre. In addition, the reduced susceptibility to Mo-MLV observed with mCAT1/insG-expressing cells correlated with impaired binding of Mo-MLV. These results show that a single amino acid insertion confers mCAT1 receptor properties on dCAT1 and provide an important insight into the co-evolution of virus-host interactions.