Phage phiC31 integrase is a recombinase that can be expressed in mammalian cells to integrate plasmids carrying an attB sequence into the genome at specific pseudo attP locations. We show by immunofluoresence that wild-type phiC31 integrase is cytoplasmic and that addition of the SV40 nuclear localization signal (NLS) localized phiC31 integrase to the nucleus. Unexpectedly, the NLS depressed integration efficiency in HeLa cells and provided no benefit when used to integrate the human Factor IX (hFIX) gene into mouse liver. As breakdown of the nuclear membrane during mitosis could allow cytoplasmic integrase access to the chromosomes, we analyzed whether cell division was required for integration into liver cells in vivo. Hepatocytes were labeled with iododeoxyuridine to mark cells that underwent DNA replication during the week after hydrodynamic injection. Hydrodynamic delivery led to DNA replication in one-third of hepatocytes. Approximately three out of four cells having phiC31 integrase-mediated stable hFIX expression did not undergo replication, indicating that cell division was not required for integrase function in liver. Therefore, although the bulk of phiC31 integrase protein seems to be cytoplasmic in mammalian cells, integration can still occur in the nucleus, even without cell division.