Abstract ATP-RNA adenylyltransferase was isolated from the 80,000 x g pellet of Pseudomonas putida. The enzyme specifically catalyzed the incorporation of AMP from ATP into a polymeric product. The Km for ATP was 3 x 10-4 m. The rate of incorporation of CMP from CTP was about 7% that of AMP, while GTP, UTP, dATP, and ADP were not utilized by the enzyme. The reaction was dependent upon the cofactor, magnesium ion (Km = 3 x 10-2 m), which could neither be replaced nor supplemented by manganese ion. The polymerization reaction, which was accompanied by the stoichiometric release of inorganic pyrophosphate, was dependent upon exogenous RNA. This requirement could be satisfied by ribosomal RNA, soluble RNA, poly C, and (Ap)3A while DNA, poly A, poly I, poly U, (Ap)2A, ApA, and (Ap)4 were ineffective. The Km for ribosomal RNA was 6 x 10-9 m and the Km for soluble RNA was 1 x 10-6 m. Ribosomal RNA functioned in the reaction by acting as a primer upon which adenylate residues were added. The polymeric product was shown to be a chain of adenylate residues, greater than 100 nucleotides in length, covalently attached to the ribosomal RNA. In addition to its role as a primer in the reaction, ribosomal RNA also appeared to act as a kinetic effector.