Abstract: An RNA ligase previously detected in extracts of <i>Escherichia coli</i> is capable of joining <i>Saccharomyces cerevisiae</i> tRNA splicing intermediates in the absence of ATP to form a 2′-5′ phosphodiester linkage (Greer, C., Javor, B., and Abelson, J. (1983) <i>Cell</i> 33, 899-906). This enzyme specifically ligates tRNA half-molecules containing nucleoside base modifications and shows a preference among different tRNA species. In order to investigate the function of this enzyme in RNA metabolism, the ligase was purified to homogeneity from <i>E. coli</i> lysate utilizing chromatographic techniques and separation of proteins by SDS-polyacrylamide gel electrophoresis. A single polypeptide of approximately 20 kilodaltons exhibited RNA ligase activity. The amino terminus of this protein was sequenced, and the open reading frame (ORF) encoding it was identified by a data base search. This ORF, which encodes a novel protein with a predicted molecular mass of 19.9 kDa, was amplified from <i>E. coli</i> genomic DNA and cloned. ORFs coding for highly similar proteins were detected in <i>Methanococcus jannaschii</i> and <i>Bacillus stearothermophilus</i>. The chromosomal gene encoding RNA ligase in <i>E. coli</i> was disrupted, abolishing ligase activity in cell lysates. Cells lacking ligase activity grew normally under laboratory conditions. However, moderate overexpression of the ligase protein led to slower growth rates and a temperature-sensitive phenotype in both wild-type and RNA ligase knockout strains. The RNA ligase reaction was studied <i>in vitro</i> using purified enzyme and was found to be reversible, indicating that this enzyme may perform cleavage or ligation <i>in vivo</i>.