We have investigated the mechanisms by which alleles at the mouse Fv-1 locus restrict replication of murine leukemia viruses. Inhibition of productive infection is closely paralleled by reduced accumulation of integrated proviral DNA as well as by reduced levels of linear viral DNA in a cytoplasmic fraction. Nevertheless, viral DNA is present at nearly normal levels in a nuclear fraction, and total amounts of viral DNA are only mildly affected in restrictive infections, suggesting a block in integration to account for reduced levels of proviral DNA. However, integrase (IN)-dependent trimming of 3' ends of viral DNA occurs normally in vivo during restrictive infections, demonstrating that not all IN-mediated events are prevented in vivo. Furthermore, viral integration complexes present in nuclear extracts of infected restrictive cells are fully competent to integrate their DNA into a heterologous target in vitro. Thus, the Fv-1-dependent activity that restricts integration in vivo may be lost in vitro; alternatively, Fv-1 restriction may prevent a step required for integration in vivo that is bypassed in vitro.