Schizosaccharomyces pombe Pfh1p is an essential member of the Pif family of 5'-3' DNA helicases. The two Saccharomyces cerevisiae homologs, Pif1p and Rrm3p, function in nuclear DNA replication, telomere length regulation, and mitochondrial genome integrity. We demonstrate here the existence of multiple Pfh1p isoforms that localized to either nuclei or mitochondria. The catalytic activity of Pfh1p was essential in both cellular compartments. The absence of nuclear Pfh1p resulted in G(2) arrest and accumulation of DNA damage foci, a finding suggestive of an essential role in DNA replication. Exogenous DNA damage resulted in localization of Pfh1p to DNA damage foci, suggesting that nuclear Pfh1p also functions in DNA repair. The absence of mitochondrial Pfh1p caused rapid depletion of mitochondrial DNA. Despite localization to nuclei and mitochondria in S. pombe, neither of the S. cerevisiae homologs, nor human PIF1, suppressed the lethality of pfh1Delta cells. However, the essential nuclear function of Pfh1p could be supplied by Rrm3p. Expression of Rrm3p suppressed the accumulation of DNA damage foci but not the hydroxyurea sensitivity of cells depleted of nuclear Pfh1p. Together, these data demonstrate that Pfh1p has essential roles in the replication of both nuclear and mitochondrial DNA.