A biochemical characterization was performed with a partially purified RNA ligase from isolated mitochondria of Leishmania tarentolae. This ligase has a K(m) of 25 ± 0.75 nM and a V(max) of 1.0 x 10-4 ± 2.4 x 10-4 nmol/min when ligating a nicked double-stranded RNA substrate. Ligation was negatively affected by a gap between the donor and acceptor nucleotides. The catalytic efficiency of the circularization of a single-stranded substrate was 5-fold less than that of the ligation of a nicked substrate. These properties of the mitochondrial RNA ligase are consistent with an expected in vivo role in the process of uridine insertion]deletion RNA editing, in which the mRNA cleavage fragments are bridged by a cognate guide RNA.