Mitochondrial biogenesis consists of the sum of all processes required for the formation of the mitochondrial membranes as well as the soluble compartments they contain. Furthermore, it includes the replication of the mitochondrial genome and correct segregation of the organelles during cell division. Mitochondrial proteins come from two sources, a limited but essential set of inner membrane proteins is encoded by the mitochondrial genome, whereas the large majority (90-95%) is derived from nucleus-encoded genes and are posttranslationally imported into the organelle. Trypanosomatids belong to the earliest diverging branches of the eukaryotic evolutionary tree which have mitochondria. This is reflected in the organisation of their mitochondrial DNA that consists of a network of two classes of topologically interlocked circular DNA molecules as well as many unique features in their mitochondrial biogenesis. The proteins encoded on the mitochondrial genome are conventional for a mitochondrial genome, their expression, however, involves a complex series of processes. Many genes represent incomplete open reading frames and their primary transcripts have to remodelled by RNA editing to convert them into translatable mRNAs. RNA editing is mediated by small mitochondria-encoded transcripts, the guide RNAs, and is in that form specific for trypanosomatids and closely related organisms. Mitochondrial translation is also unconventional. No tRNA genes are encoded on the mitochondrial genome. Instead, mitochondrial protein synthesis functions exclusively with imported cytosolic, eukaryotic-type tRNAs. The composition of mitochondrial ribosomes is also unusual in that they contain the smallest known rRNAs. They are about 30% shorter than the already much reduced rRNAs in human mitochondria. Furthermore, the topological organisation of the mitochondrial genome requires an elaborate replication machinery involving topoisomerases. Finally, some trypanosomatids have life cycle stages exhibiting very different mitochondrial activities and can therefore serve as a model system for the regulation of mitochondrial biogenesis.