We describe the purification, cloning, and characterization of the CCA-adding enzyme [ATP(CTP):tRNA nucleotidyl transferase] from the thermophilic archaebacterium, Sulfolobus shibatae. Characterization of an archaeal CCA-adding enzyme provides formal proof that the CCA-adding activity is present in all three contemporary kingdoms. Antibodies raised against recombinant, expressed Sulfolobus CCA-adding enzyme reacted specifically with the 48-kDa protein and fully depleted all CCA-adding activity from S. shibatae crude extract. Thus, the cloned cca gene encodes the only CCA-adding activity in S. shibatae. Remarkably, the archaeal CCA-adding enzyme exhibits no strong homology to either the eubacterial or eukaryotic CCA-adding enzymes. Nonetheless, it does possess the active site signature G[SG][LIVMFY]xR[GQ]x5,6D[LIVM][CLIVMFY]3-5 of the nucleotidyltransferase superfamily identified by Holm and Sander (1995, Trends Biochem Sci 20:345-347) and sequence comparisons show that all known CCA-adding enzymes and poly(A) polymerases are contained within this superfamily. Moreover, we propose that the superfamily can now be divided into two (and possibly three) subfamilies: class I, which contains the archaeal CCA-adding enzyme, eukaryotic poly(A) polymerases, and DNA polymerase beta; class II, which contains eubacterial and eukaryotic CCA-adding enzymes, and eubacterial poly(A) polymerases; and possibly a third class containing eubacterial polynucleotide phosphorylases. One implication of these data is that there may have been intraconversion of CCA-adding and poly(A) polymerase activities early in evolution.