Genetic subtyping of feline immunodeficiency virus (FIV) was carried out by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. A 329-base pair fragment in the FIV gag gene was amplified by nested PCR, then digested with restriction enzymes, HindIII, PvuII and BamHI. Using these restriction enzymes, FIV isolates belonging to subtypes A, B and D, which had been classified on the basis of the env gene V3-V5 sequence, could be discriminated. Genetic subtypes of FIV prevalent in Japan were investigated using the gag-nested PCR-RFLP analysis. Of 88 FIV-infected cats, PCR products of 70 cats showed a subtype B RFLP pattern (digestion only with PvuII), those of nine cats had a subtype D RFLP pattern (digestion only with BamHI), and those of seven cats had a subtype A RFLP pattern (digestion only with HindIII). The PCR products of the remaining two cats had subtype A and B RFLP patterns (digestion with both HindIII and PvuII). The digestion pattern of the gag-nested PCR-RFLP analysis was unchanged after in vivo passages of the virus. These results suggest that the gag-nested PCR-RFLP analysis is useful as a simple method for FIV genetic subtyping.