Ligase activity was detected in extracts of Escherichia coli, Clostridium tartarivorum, Rhodospirillum salexigens, Chromatium gracile, and Chlorobium limicola. Ligase was measured by joining of tRNA halves produced from yeast IVS-containing tRNA precursors by a yeast endonuclease. The structure of tRNA^(Tyr) halves joined by an E. coli extract was examined. The ligated junction is resistant to nuclease P1 and RNAase T2 but sensitive to venom phosphodiesterase and alkaline hydrolysis, consistent with a 2′,5′ linkage. The nuclease-resistant junction dinucleotide comigrates with authentic (2′,5′) A^PA marker in thin-layer chromatography. The phosphate in the newly formed phosphodiester bond is derived from the pre-tRNA substrate. The widespread existence of a bacterial ligase raises the possibility of a novel class of RNA processing reactions.