A difference between prokaryotic RNase HII and HIII, which both belong to type 2 RNase H, is a long N-terminal extension of HIII; however, the main-fold structures of HII and HIII known as RNase H-fold are similar. To further understand the structure-function relationship of RNase HII and RNase HIII, biochemical analyses were carried out using N-terminal truncations of RNase HIII (IIIN56(Δ), IIIN81(Δ), and IIIN88(Δ)) and C-terminal truncation (IIC19(Δ)) of RNase HII from Chlamydophila pneumoniae. Compared with wild-type CpRNase HII/III, IIIN56(Δ) had no obvious variation on the cleavage site and efficiency of DNA-rN(1)-DNA/DNA (DR(1)D) and DNA-rN(4)-DNA/DNA (DR(4)D) substrates. IIC19(Δ) and IIIN81(Δ) both showed decreased activities, and IIIN88(Δ) exhibited little cleavage on these substrates. However, IIIN81(Δ) showed very different activities toward different substrates (20% for DR(1)D and 85% for DR(4)D). Moreover, IIC19(Δ)IIIN(82-88) mutant, prepared through adding N-terminal 82nd to 88th residues locating at the bound region of N- and C-terminal domains of CpRNase HIII to N-terminus of IIC19(Δ), cleaved DR(4)D substrate more efficiently and preferentially at the cleavage sites of CpRNase HIII but not those of CpRNase HII. These results indicated that C-termini of CpRNase HII, N-termini of CpRNase HIII, and bound region of N- and C-terminal domain are all important for enzymatic activities. Moreover, the 82nd to 88th residues of N-terminus of CpRNase HII are related with enzyme cleavage site specificity. These results will help to understand the importance of C-termini of CpRNase HII and N-termini of CpRNase HIII to the enzyme activities for DR(1)D and DR(4)D substrate.