Intron removal from tRNA precursors involves cleavage by a tRNA splicing endonuclease to yield tRNA 3'-halves beginning with a 5'-hydroxyl, and 5'-halves ending in a 2',3'-cyclic phosphate. A tRNA ligase then incorporates this phosphate into the internucleotide bond that joins the two halves. Although this 3'-P RNA splicing ligase activity was detected almost three decades ago in extracts from animal and later archaeal cells, the protein responsible was not yet identified. Here we report the purification of this ligase from Methanopyrus kandleri cells, and its assignment to the still uncharacterized RtcB protein family. Studies with recombinant Pyrobaculum aerophilum RtcB showed that the enzyme is able to join spliced tRNA halves to mature-sized tRNAs where the joining phosphodiester linkage contains the phosphate originally present in the 2',3'-cyclic phosphate. The data confirm RtcB as the archaeal RNA 3'-P ligase. Structural genomics efforts previously yielded a crystal structure of the Pyrococcus horikoshii RtcB protein containing a new protein fold and a conserved putative Zn(2+) binding cleft. This structure guided our mutational analysis of the P. aerophilum enzyme. Mutations of highly conserved residues in the cleft (C100A, H205A, H236A) rendered the enzyme inactive suggesting these residues to be part of the active site of the P. aerophilum ligase. There is no significant sequence similarity between the active sites of P. aerophilum ligase and that of T4 RNA ligase, nor ligases from plants and fungi. RtcB sequence conservation in archaea and in eukaryotes implicates eukaryotic RtcB as the long-sought animal 3'-P RNA ligase.