Endonuclease inhibitor was isolated from the chloroplast of the leaves of tobacco, Burley 21 and NC 83, The inhibitory effect on the activities of endonucleases including BamH1 was lost by the addition of trypsin or heat treatment However, the treatment of $alpha$-amylase was unconcerned in the inhibitory effect The endonuclease inhibitor was found to be a monomeric protein that plays a vital role for protection of tobacco chloroplast DNA from endonuclease action.