It has been suggested that FV3 DNA replication occurs in two stages [R. Goorha (1982) J. Virol. 43, 519-528]. First-stage DNA synthesis is restricted to the nucleus, where the replicating DNA ranges from genome to twice genome size; second-stage DNA replication occurs exclusively in the cytoplasm, and the replicating DNA is concatameric. A temperature-sensitive mutant (ts 12488) of FV3, at a nonpermissive temperature (30 degrees), synthesized DNA in the nucleus only, and the size of the replicative complex (as determined by neutral sucrose gradient analysis) was between genome and twice genome length. These characteristics establish that at nonpermissive temperature, ts 12488 is arrested in the first stage of DNA replication. Temperature shift-down (30 degrees----25 degrees) of ts 12488-infected cells at 4 hr postinfection showed that, within 30 min of the shift, the replicative complex became very large (more than 10 times genome size). Furthermore, newly synthesized DNA was now found in the cytoplasmic fraction also. These results suggest that ts 12488, upon shift-down, enters into the second stage of DNA replication where progeny DNA is synthesized as a large concatamer. In shift-down experiments, de novo protein synthesis was not required to initiate second-stage DNA replication, strongly suggesting that the thermosensitive protein is directly involved in second-stage DNA replication. This genetic evidence establishes the previous biochemical findings of a two-stage replication scheme for FV3 DNA.