The Japanese isolate (TM1 strain) of feline immunodeficiency virus (FIV) which replicates in a feline CD4 (fCD4)-positive lymphoblastoid cell line (MYA-1 cells) was molecularly cloned from extrachromosomal closed circular DNA. The restriction map of the clone, termed pFTM 191 complete genome (CG), showed a considerable difference from that of the U.S. isolate (Petaluma strain) of FIV. The sequence homology in the long terminal repeat between the TM1 and Petaluma strain was 82%. The pFTM 191 CG was biologically active after transfection into Crandell feline kidney cells which were permissive for replication of FIV Petaluma. However, the progeny virions could not reinfect fCD4-negative Crandell feline kidney cells but could infect fCD4-positive MYA-1 cells. When a specific-pathogen-free cat was inoculated with the virus derived from the pFTM 191 CG, the cat seroconverted within 8 weeks postinoculation and FIV was reisolated at 4, 8, and 20 weeks postinoculation. These results indicate the infectivity of the pFTM 191 CG in vivo.