We have identified a novel polymerase beta (Pol beta)-like enzyme from Leishmania infantum, a parasite protozoon causing disease in humans. This protein, named Li Pol beta, shows a nuclear localization that contrasts with the mitochondrial localization of Pol beta from Crithidia fasciculata, a closely related parasite, the only polymerase beta described so far in Trypanosomatidae. Li Pol beta, that belongs to the DNA polymerase X family, displays an evolutionarily conserved Pol beta-type DNA polymerase core, in which most of the key residues involved in DNA binding, nucleotide binding, dRPase and polymerization catalysis are conserved. In agreement with this, Li Pol beta, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity. Cell synchronization experiments showed a correlation between both Li Pol beta mRNA and protein levels along the parasite cell cycle. Analysis of these parameters at the different growth phases of the parasite, from the proliferative (non-infective) logarithmic phase to the non-dividing (highly infectious) stationary phase, showed high levels of Li Pol beta at the infective phase of the parasite. The data suggest a role of Li Pol beta in base excision repair in L.infantum, a parasite usually affected by oxygen stress environments into the macrophage host cells.