Abstract An RNA phosphodiesterase of molecular weight about 100,000 was purified 330-fold from the cell-free culture fluid of a Bacillus species grown on RNA as the sole source of phosphorus. In the presence of Mg2+ or Mn2+, this nuclease catalyzes hydrolysis of high molecular weight RNA and synthetic polyribonucleotides, but not of DNA, dinucleoside monophosphates, 2',3'- or 3',5'-cyclic nucleotides or adenylate oligonucleotides terminating in a 2',3'-cyclic adenylic acid residue. While the initial products are nucleoside 5'-phosphates, nucleosides eventually are formed due to the presence of a 5'-nucleotidase activity which purifies together with the phosphodiesterase. These two activities, which are not separable by acrylamide gel electrophoresis or by sucrose density gradient centrifugation, account for the ability of the organism to grow on RNA as a sole source of phosphorus. Polymer cleavage appears to proceed from the 3'-hydroxyl terminus and to be exonucleolytic. Oligonucleotides characteristic of endonucleolytic degradation are not formed as intermediates during hydrolysis of RNA, and ApCpC and CpCpA are hydrolyzed to ApC plus cytidine and CpC plus adenosine, respectively. Ribose- or deoxyribose-nucleoside 5'-phosphates, but not nucleoside 2'- or 3'-phosphates, are good substrates for the 5'-nucleotidase activity.