Endonuclease IV has AP endonuclease and 3'-repair diesterase activities. Here, we report Chlamydophila pneumoniae endonuclease IV (CpEndoIV) could hydrolyze the ds DNA and the RNA strand of RNA/DNA hybrid from the 3' end, yet the DNA strand of RNA/DNA hybrid was not the effective substrate of CpEndoIV. The optimal pH for 3' exonuclease on double-stranded (ds) DNA and RNA/DNA hybrids were both basic, but with some difference. The effect of divalent ions (Mg(2+), Ca(2+), Zn(2+), Cu(2+), Ni(2+), and Mn(2+)) on 3' exonuclease was different for both substrates. High concentration of NaCl inhibited 3' exonuclease on both substrates. For both substrates, the 3' exonuclease activity of CpEndoIV on matched and mismatched 3' end was comparable.