By using a focus reduction assay in CrFK feline fibroblast cells, virus neutralizing antibodies (VNA) to feline immunodeficiency virus (FIV) were demonstrated in cats that had been naturally or experimentally infected with FIV. The antigenic relatedness of four strains of FIV, divergent in nucleotide sequence within the env gene, was investigated by neutralization following adaptation of each virus for growth in CrFK cells. Two of the viruses were from The Netherlands (FIV/AM-4 and AM-6), one was from the U.K. (FIV/GL-8) and one was from the U.S.A. (FIV/PET). Reaction of the viruses in the neutralization assay with cat antibodies to homologous or heterologous strains indicated that while there was a degree of cross-reactivity between all four, there were consistent differences suggesting the existence of FIV neutralization subtypes. In particular, FIV/PET and FIV/AM-6 were closely related but FIV/PET and FIV/GL-8 were clearly distinct. VNA from naturally infected cats in the field showed a pattern of reactivity against FIV/PET and FIV/GL-8 that confirmed the antigenic diversity of FIV.