We have studied the question of whether a retrovirus integrates into the chromosomal DNA of the host cell before or after the DNA is replicated during the S phase of the cell cycle. We have infected single NIH-3T3 cells with BAG, a replication-incompetent retroviral vector which encodes the lacZ gene, then observed the clones derived from these cells to discover whether all the cells carry a copy of the proviral DNA. We have discovered that only half of the progeny of an infected cell carries a copy of the provirus. This indicates that the virus only integrates into post-replication DNA. We discuss the implications of this result for applications of retroviruses, such as gene therapy and cell lineage, which use them as vehicles for gene transfer into stem cells.