Full-length cDNAs for DNA ligase IV and the alpha and beta isoforms of DNA ligase III were cloned from Xenopus laevis to permit study of the genes encoding mitochondrial DNA ligase. DNA ligase III alpha and III beta share a common NH(2) terminus that encodes a mitochondrial localization signal capable of targeting green fluorescent protein to mitochondria while the NH(2) terminus of DNA ligase IV does not. Reverse transcriptase-polymerase chain reaction analyses with adult frog tissues demonstrate that while DNA ligase III alpha and DNA ligase IV are ubiquitously expressed, DNA ligase III beta expression is restricted to testis and ovary. Mitochondrial lysates from X. laevis oocytes contain both DNA ligase III alpha and III beta but no detectable DNA ligase IV. Gel filtration, sedimentation, native gel electrophoresis, and in vitro cross-linking experiments demonstrate that mtDNA ligase III alpha exists as a high molecular weight complex. We discuss the possibility that DNA ligase III alpha exists in mitochondria in association with novel mitochondrial protein partners or as a homodimer.