The human DNA polymerase lambda (Polλ) is a DNA repair polymerase, which is believed not only to play a role in base excision repair but also to contribute to DNA double-strand break repair by non-homologous end joining. We described here that cellular expression of the recently described natural polymorphic variant of Polλ, Polλ(R438W), affects the homologous recombination (HR) pathway and sister chromatid exchange (SCE) events. We show that the HR defect provoked by this polymorphism enhances cellular sensitivity to the anticancer agent camptothecin (CPT), most of whose DNA damage is repaired by HR. All these effects were dependent on the DNA polymerase activity of Polλ(R438W) as the expression of a catalytically inactive Polλ(R438W) did not affect either the HR and SCE frequencies or the cellular sensitivity to CPT. These results suggest that sensitivity to CPT could result from cancer-related mutation in specialized DNA repair polymerases.