Iron–sulphur clusters (ISCs) are protein co-factors essential for a wide range of cellular functions. The core iron–sulphur cluster assembly machinery resides in the mitochondrion, yet due to export of an essential precursor from the organelle, it is also needed for cytosolic and nuclear iron–sulphur cluster assembly. In mitochondria all [4Fe–4S] iron–sulphur clusters are synthesised and transferred to specific apoproteins by so-called iron–sulphur cluster targeting factors. One of these factors is the universally present mitochondrial Nfu1, which in humans is required for the proper assembly of a subset of mitochondrial [4Fe–4S] proteins. Although most eukaryotes harbour a single Nfu1, the genomes of Trypanosoma brucei and related flagellates encode three Nfu genes. All three Nfu proteins localise to the mitochondrion in the procyclic form of T. brucei, and TbNfu2 and TbNfu3 are both individually essential for growth in bloodstream and procyclic forms, suggesting highly specific functions for each of these proteins in the trypanosome cell. Moreover, these two proteins are functional in the iron–sulphur cluster assembly in a heterologous system and rescue the growth defect of a yeast deletion mutant.