Despite many years of research translation in human mitochondria is still poorly understood. Up to now only a few factors involved in this process have been identified. The aim of this research is to characterise the protein involved in mitochondrial ribosome recycling after translation termination has been completed. Although a candidate protein was identified bioinformatically on the basis of sequence similarity, no further investigations had been performed. We have subsequently cloned this gene, namely mitochondrial ribosome recycling factor (mtRRF). A series of experiments have been designed to determine whether mtRRF is truly mitochondrial in vivo. i) A GFP fusion construct was generated and HeLa cells transiently transfected. Mitochondria were visualised by Mitotracker staining and positive co-localisation was identified microscopically. ii) To confirm that this co-localisation truly reflected internalisation into mitochondria we performed an in vitro import assay into isolated organelles. Bacterial RRF has been described as an essential protein. To determine if the same is true for the mitochondrial counterpart, the steady-state levels have been downregulated by the application of siRNA in HeLa cells. Transfected cells develop morphological changes and loss of mtRRF for more than 6 days appears lethal. Thus is appears that mtRRF is also an essential gene for higher eukaryotic cells, consistent with a critical role in mitochondrial function.