Human CD4, the receptor for the gp120 envelope glycoprotein of HIV-1, is the route for viral entry into CD4+ cells; other cellular factors may cooperate with CD4 to facilitate HIV-1 entry into human cells. Human CD4 expressed on murine cells does not readily mediate HIV-1 entry, which may reflect a functional incompatibility of human CD4 with murine cellular components. We postulated that a HIV-1 gp120-binding mutant murine CD4 (L3T4) possessing a minimal number of human amino acid residues could facilitate HIV-1 entry into rodent cells, unlike human CD4. This hypothesis led us to develop a series of murine L3T4 mutants that bear human CD4 gp120-binding region amino acid residues while retaining most L3T4 epitopes. HeLa cell transfectants expressing gp120-binding mutant L3T4 proteins could be infected with HIV-1. Three mouse cell lines expressing these L3T4 mutant proteins could also be infected with HIV-1 as determined by PCR techniques that detect viral DNA and spliced RNAs. Lectin-stimulated polymorphonuclear leukocytes from transgenic mice (SBL mouse) expressing a gp120-binding L3T4 mutant protein were infected with HIV-1 at the same frequency as lectin-stimulated human peripheral blood lymphocytes as determined by in situ PCR analyses. Supernatant p24gag and reverse transcriptase levels in HIV-infected mouse cell cultures, however, were routinely at background levels, unlike HIV-infected human cell cultures. Thus, gp120-binding mutant L3T4 proteins mediate viral entry in all mouse cells that were tested, but high-level viral replication is absent in these cells.