A number of factors affect the infectivity of retroviruses. The effect of pH on infectivity and morphology of ecotropic moloney murine leukemia virus (MoMuLV) was determined in this work. The ecotropic MoMuLVs were found to remain infectious at a narrow pH range from 5.5 to 8.0. Our experiments indicated that the viruses were inactivated swiftly at lower or higher pH. Within 5 min of exposure to pH 4 about 95% of the viruses lost infectiousness. The viruses were completely inactivated after exposure to pH < 3 or pH >11 for 5 min. The inactivation of MoMuLV was irreversible. Electron microscopy revealed that ecotropic MoMuLV remained round-shaped at pH between 7.0 and 5. They became irregular with a convex head at pH < 4. At pH 2, virtually all virion particles were penetrated by stains, causing the accumulation of heavy metals inside the particles. The penetration of heavy metal inside the particles indicated the disassociation of the lipid bilayer of the viruses at low pH. A FACS-based screening strategy for selecting high-titer retrovirus producing cell lines is also presented in this report.