Procedures have been developed for the facile preparation of wheat seedling nuclease in highly purified form. The preparation appears to be homogeneous by many physical criteria; however, analytical gel electrophoresis reveals 12 protein bands, only one of which is catalytically active. It is suggested that most of the inactive species are artifacts formed from active enzyme by processes which increase the negative charge of the protein. The enzyme rapidly catalyzes the hydrolysis of denatured DNA and RNA to acid-soluble products and also of the 3'-phosphomonoester linkage of a variety of 3'-mononucleotides. The enzyme has very little, if any, activity toward native DNA with respect to the production of acid-soluble substances; however, athe succeeding paper demonstrates that native DNA is cleaved at a few specific loci to yield large duplex DNA fragments. The nuclease has been characterized as having endonucleolytic activity towards denatured DNA and primarily exonucleolytic activity towards RNA. The mononucleotides produced under the influence of the enzyme bear 5'-phosphomonoester groups. Various lines of evidence indicate a relatively high preference of the enzyme for the hydrolysis primarily of 3'-phosphoester linkages of adenylic acid units and secondarily of thymidylic or uridylic acid units in DNA and in ribohomopolymers, respectively. Corresponding linkages involving cytidylic acid and especially guanylic acid are relatively resistant. The 3'-nucleotidase activity of the enzyme at pH 5.0 towards the various mononucleotides fits the same pattern; i.e. nucleotides containing adenine are hydrolyzed most rapidly, followed in decreasing order by those containing thymine or uracil, cytosine, and guanine.