Full-length cDNAs for DNA ligase IV and the a and b isoforms of DNA ligase III were cloned from Xenopus laevis to permit study of the genes encoding mitochondrial DNA ligase. DNA ligase IIIa and IIIb share a common N-terminus that encodes a mitochondrial localization signal capable of targeting green fluorescent protein to mitochondria while the N-terminus of DNA ligase IV does not. RT-PCR analyses with adult frog tissues demonstrate that while DNA ligase IIIa and DNA ligase IV are ubiquitously expressed, DNA ligase IIIb expression is restricted to testis and ovary. Mitochondrial lysates from Xenopus laevis oocytes contain both DNA ligase IIIa and IIIb but no detectable DNA ligase IV. Gel filtration, sedimentation, native gel electrophoresis and in vitro cross-linking experiments demonstrate that mtDNA ligase IIIa exists as a high molecular weight complex. We discuss the possibility that DNA ligase IIIa exists in mitochondria in association with novel mitochondrial protein partners or as a homodimer.