Biohydrogen is considered as an important key to a sustainable world power supply and is currently being seen as the versatile fuel of the future, with the potential to replace fossil fuels. The most efficient biohydrogen producers are hydrogenases. Nevertheless, due to the complex maturation processes of these enzymes, their heterologous production leaves some intriguing points not elucidated up to now. The limit of our understanding in this field makes a barrier for hydrogenases application in a variety of biotechnological processes. This review focuses on recent progresses in the development of heterologous production systems and cell-free maturation systems for the biosynthesis of active [Fe-Fe] and [Ni-Fe] hydrogenases. It also highlights some up to now un-discussed questions on the probable existence of unknown machinery able to maturate [Fe-Fe] hydrogenases or a contribution of the [Ni-Fe] hydrogenases maturases to the formation of an active H-cluster for the [Fe-Fe] hydrogenases. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.