The Fv-1 gene of the mouse is a cellular gene, which prevents the replication of murine leukemia viruses. We investigated its intracellular site of action and found that the Fv-1 gene product blocks the appearance of virus-specific RNA in nonpermissive cells, which suggested a transcriptional or a preintegration block. Experiments performed to distinguish between these two possibilities revealed that the Fv-I gene restriction prevents integration of the reverse transcribed proviral DNA into the cell genome. However, we also found that the levels of unintegrated proviral DNA were not significantly affected by the Fv-1 gene restriction soon after infection. These data show that the Fv-1 gene product inhibits a step of the virus life cycle after the synthesis of proviral DNA but before its integration.