Replication of kDNA in the mitochondrion of the kinetoplastid protozoan is an essential process. One of the proteins that may be required for the kDNA replication is the ribonuclease H (RNase H; EC 3.1.26.4). We have identified four distinct ribonuclease H genes in Leishmania, one type I (LRNase HI) and three type II (LRNase HIIA, LRNase HIIB and LRNase HIIC). We detail here molecular characterization of LRNase HIIC. The coding sequence of LRNase HIIC is 1425 bp in length encoding a 474-amino acid protein with a calculated molecular mass of approximately 53 kDa. While LRNase HIIC shares several conserved domains with mitochondrial RNase H from other organisms, it has three extra patches of amino acid sequences unique to this enzyme. Functional identity of this protein as an RNase H was verified by genetic complementation in RNase H-deficient Escherichia coli. The precursor protein may be enzymatically inactive as it failed to complement the E. coli mutant. The mitochondrial localization signal in LRNase HIIC is within the first 40 amino acid residues at the N-terminus. In vitro import of the protein by the mitochondrial vesicles showed that the precursor protein is processed to a 49-kDa protein. Antisense ablation of LRNase HIIC gene expression is lethal to the parasite cells both in vitro and in vivo. This study not only reveals the significance of the LRNase HIIC in the kinetoplast biology but also identifies a potential molecular target for antileishmanial chemotherapy.