We have investigated targeting of retroviral vectors to a mutant EGF receptor (EGFRvIII) that is expressed in cancers of the brain, breast, lung and ovary, but is not found in any normal tissues. An expression plasmid was made in which a single chain Fv antibody specific for EGFRvIII was inserted at a novel position within a disulphide-bonded surface loop near the native receptor binding site of the Moloney leukemia virus ecotropic envelope glycoprotein. This fusion protein was expressed and incorporated into retroviral particles as efficiently as normal envelope glycoprotein. Retroviral vectors made with the fusion protein were able to bind peptide antigen and EGFRvIII expressed on the surface of human glioblastoma cells. The retroviral vectors had normal levels of infectivity on mouse cells, showing that the envelope glycoprotein tolerated a large insertion at this site, but did not show significant infectivity to human cells expressing EGFRvIII. Thus we were able to redirect retrovirus binding to this tumour-specific target without perturbing the normal function of the ecotropic envelope glycoprotein, but this was not sufficient to mediate infectivity via this receptor.