Purified lupine tRNA nucleotidyltransferase catalyzed only the incorporation of AMP and CMP into 3'-terminal-C-C-A sequences of tRNA as determined by terminal analysis of the reaction product. The incorporation of AMP and CMP was inhibited by their respective triphosphates to different degrees; UTP also showed an inhibitory effect. The pH optimum of the purified enzyme was found to be 9.5 in glycine buffer. The enzyme required magnesium or manganese ions for its activity.-SH reagents reversibly inhibited the action of the enzyme. Kinetic data, the different effects of ionic strength on the incorporation of AMP and CMP, and different rates of thermal inactivation for AMP and CMP incorporation in connection with chromatographic properties of the enzyme suggest the existence of only one form of tRNA nucleotidyltransferase with different catalytic sites for ATP and CTP.