Iron-sulfur [Fe-S] clusters are ubiquitous ancient prosthetic groups that are required to sustain fundamental life processes. Formation of intracellular [Fe-S] clusters does not occur spontaneously but requires a complex biosynthetic machinery. Different types of [Fe-S] cluster assembly systems have been discovered. All of them have in common the requirement of a cysteine desulfurase and the participation of [Fe-S] scaffold proteins. The purpose of this review is to discuss various aspects of the molecular mechanisms of [Fe-S] cluster assembly in living organisms: (i) mechanism of sulfur donor enzymes, namely the cysteine desulfurases; (ii) mechanism by which clusters are preassembled on scaffold proteins and (iii) mechanism of [Fe-S] cluster transfer from scaffold to target proteins.