Adenylyl (cytidylyl)-tRNA nucleotidyltransferase (ATP (CTP): tRNA adenylyl (cytidylyl)transferase, EC2.7.7.25) has been purified 11,800-fold from a crude extract of Escherichia coli B in an overall yield of 23%. The key step in this purification is the use of a tRNA-Sepharose affinity column. The purified enzyme has a specific activity of approximately 280 mumol of AMP incorporated/min/mg of protein at 37 degrees and has a molecular weight of 52,000 as determined by sodium dodecyl sulfate gel electrophoresis of Sephadex chromatography. The turnover number of the pure enzyme, under optimal assay conditions, is estimated as 21,000, and we believe it constitutes only o.oo6% of the total cellular protein. Both AMP- and CMP-incorporating activities have an identical isoelectric point of 5.85. The AMP-incorporating activity of the enzyme is inhibitied by some transition metal chelating agents but not by others.