HLTF is highly similar in domain organisation to yeast Rad5. We identify PTIP and RPA70, both involved in DNA replication and DNA repair, as HLTF-interacting proteins although cells depleted of HLTF did not show defects in cellular responses to DNA damage. In vitro, HLTF has ATPase activity and E3 ubiquitin ligase activity with a range of E2 ubiquitin conjugating enzymes. HLTF expression is severely reduced in a range of cancer cells, and we suggest that the HLTF antibodies generated in this study could be used for cancer diagnostic purposes.