We have engineered ecotropic Moloney murine leukemia virus-derived envelopes targeted to cell surface molecules expressed on human cells by the N-terminal insertion of polypeptides able to bind either Ram-1 phosphate transporter (the first 208 amino acids of amphotropic murine leukemia virus surface protein) or epidermal growth factor receptor (EGFR) (the 53 amino acids of EGF). Both envelopes were correctly processed and incorporated into viral particles. Virions carrying these envelopes could specifically bind the new cell surface receptors. Virions targeted to Ram-1 could infect human cells, although the efficiency was reduced compared with that of virions carrying wild-type amphotropic murine leukemia virus envelopes. The infectivity of virions targeted to EGFR was blocked at a postbinding step, and our results suggest that EGFR-bound virions were rapidly trafficked to lysosomes. These data suggest that retroviruses require specific properties of cell surface molecules to allow the release of viral cores into the correct cell compartment.