Over the past decade a large volume of research data has accumulated which has established a fundamental role for mitochondria in normal cellular functioning, as well as in various pathologies. Mitochondria play a pivotal role in metabolism and energy production, and are one of the key players involved in programmed cell death. On the other hand, mitochondrial dysfunction is implicated, directly or indirectly in numerous pathological conditions including inherited mitochondrial disorders, diabetes, cardiovascular and neurodegenerative diseases, and a variety of malignancies. The ability to modulate mitochondrial function by altering the diverse protein component of this organelle may be of great value for developing future therapeutic interventions. This review will discuss approaches used to introduce proteins into mitochondria. One group of methods utilizes strategies aimed at expressing proteins from genes in the nucleus. These include overexpression of nuclear-encoded mitochondrial proteins, allotopic expression, which is the re-coding and relocation of mitochondrial genes to the nucleus for expression and subsequent delivery of their gene products to mitochondria, and xenotopic expression, which is the nuclear expression of genes coding electron transport chain components from distant species, for delivery of their products to mammalian mitochondria. Additionally, antigenomic and progenomic strategies which focus on expression of mitochondrially targeted nuclear proteins involved in the maintenance of mtDNA will be discussed. The second group of methods considered will focus on attempts to use purified proteins for mitochondrial delivery. Special consideration has been given to the complexities involved in targeting exogenous proteins to mitochondria.