The development of virus neutralizing (VN) antibody is one of the most effective host defense mechanisms against virus infection. In the present study, we developed a new VN assay against feline immunodeficiency virus (FIV) using a feline T-lymphoblastoid cell line, MYA-1 cells, based on inhibition of viral reverse transcriptase production. This assay is applicable to strains of FIV which can not infect CRFK cells. By using the assay, we examined long-term responses of VN antibody in cats experimentally infected with FIV. VN antibody titers increased progressively during first 30 weeks post inoculation and remained at high titers thereafter for 7 years of observation periods.