Protein trafficking and translocation are essential processes in even the simplest living cells. The compartmentalisation within eukaryotic cells places a very high demand on the fidelity of protein trafficking and translocation, since a large percentage of the cell's protein complement is inserted into, or translocated across membranes. Indeed, most mitochondrial proteins are imported from the cytosol into the organelle and reach their final destination with the assistance of versatile translocation machineries. The first components involved in mitochondrial protein import were identified about 20 years ago and over the last two decades many new factors and machineries have been brought to light. However, in spite of these discoveries we still have much to explore regarding the molecular mechanisms that distinguish the different mitochondrial import pathways. In particular, an open question that requires deeper exploration is the role of lipids and lipid modifying enzymes in this process. Mitochondrial biogenesis requires the coordinated synthesis and import of both proteins and phospholipids, however, these have typically been considered as distinct research fields. Recent findings have placed phospholipids at the forefront of research dealing with mitochondrial biogenesis, in particular their role in the regulation of mitochondrial transport machineries. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.