Nodaviruses encode an RNA-dependent RNA polymerase called Protein A that is responsible for replication of the viral RNA segments. The intracellular localization of Protein A from a betanodavirus isolated from Atlantic halibut (AHNV) was studied in infected fish cells and in transfected mammalian cells expressing Myc-tagged wild type Protein A and mutants. In infected cells Protein A localized to cytoplasmic structures resembling mitochondria and in transfected mammalian cells the AHNV Protein A was found to co-localize with mitochondrial proteins. Two independent mitochondrial targeting signals, one N-terminal comprising residues 1-40 and one internal consisting of residues 225-246 were sufficient to target both Protein A deletion mutants and enhanced green fluorescent protein (EGFP) to the mitochondria. The N-terminal signal corresponds to the mitochondrial targeting sequence of the Flock House Virus (FHV) Protein A while the internal signal is similar to the single targeting signal previously found in Greasy Grouper Nervous Necrosis Virus (GGNNV) Protein A.