The envelope protein of retroviruses is responsible for viral entry into host cells. Here, we describe a mutational library approach to dissect functional domains of the envelope protein involving a retroviral vector, which expresses both the envelope protein of Akv murine leukemia virus (MLV) and the neomycin phosphotransferase II (Neo) selection marker from the same transcript. Envelope expression was achieved by inserting an internal ribosome entry site (IRES) between the neo and the env genes. We found the structure of the linker between the IRES element and env to be critical for sufficient envelope expression. This vector functions as a replication competent mini-virus in a culture of NIH 3T3 derived semi-packaging cells that express the viral Gag and Pol proteins. Titers comparable to those of wild type virus were achieved by this system. To test this vector system, we created a random mutational library of Arg 85 and Asp 86 in the first variable region of Akv envelope protein. Homologous amino acids to Asp 86 in Moloney and Friend murine leukemia viruses are thought to be directly involved in receptor binding. Subsequent selection of mutants capable of infecting murine NIH 3T3 cells indicated that the wild type aspartic acid or another hydrophilic residue at position 86 is an important determinant for envelope function.