DNA replication in mammalian cells occurs in discrete nuclear foci called 'replication factories'. Here we show that DNA ligase I, the main DNA ligase activity in proliferating cells, associates with the factories during S phase but displays a diffuse nucleoplasmic distribution in non-S phase nuclei. Immunolocalization analysis of both chloramphenicol acetyltransferase (CAT)-DNA ligase I fusion proteins and epitope tagged DNA ligase I mutants allowed the identification of a 13 amino acid functional nuclear localization signal (NLS) located in the N-terminal regulatory domain of the protein. Furthermore, the NLS is immediately preceded by a 115 amino acid region required for the association of the enzyme with the replication factories. We propose that in vivo the activity of DNA ligase I could be modulated through the control of its sub-nuclear compartmentalization.