DNA ligase I (Lig I) has key roles in chromosomal DNA replication and repair in the eukaryotic cell nucleus. In the budding yeast Saccharomyces cerevisiae the Lig I enzyme Cdc9p is also required for mitochondrial DNA replication and repair. In this report, dual nuclear-mitochondrial localization is demonstrated to be a property of the essential Lig I enzyme Cdc17 from the distantly related fission yeast Schizosaccharomyces pombe. Expression of nuclear and mitochondrial forms of Cdc17 from separate genes shows that, whereas expression of either protein alone is insufficient to restore viability to cells lacking endogenous Cdc17, co-expression restores full viability. In the nucleus, Lig I interacts with the sliding clamp proliferating cell nuclear antigen (PCNA) via a conserved PCNA interacting sequence motif known as a PIP box. Deletion of the PIP motif from the N-terminus of the nuclear form of Cdc17 fails to abolish Cdc17 function, indicating that PCNA binding by Cdc17 is not an absolute requirement for completion of S-phase.