Through interspecific hybridization and subsequent backcrossing, genes and genomes may be transferred over the species barrier. In Sweden, the introduced brown hare Lepus europaeus hybridizes with the native mountain hare L. timidus. To investigate the direction and the extent of transfer of mitochondrial DNA (mtDNA) between the species, the mtDNA haplotypes were screened in 522 brown hares and 149 mountain hares from areas of sympatry and allopatry. A total of 51 brown hares with mountain hare mtDNA, but no mountain hares with brown hare mtDNA were detected. Thus, mtDNA transfer over the species barrier is directed from mountain hares to brown hares. We argue that frequency-dependent hybridization and/or interspecific male competition mediates this directionality. Further, the percentage of brown hares with transmitted mountain hare mtDNA was lower in areas of former species sympatry (0.6%) compared to areas of current sympatry (15%). Thus, the transferred mtDNA may disappear from brown hare populations if there is no continuous input through hybridization. We suggest that specimens with an alien mtDNA experience a fitness reduction as a result of a functional incompatibility between the cytoplasmic mitochondrial genomes and the cell nucleus.