In recent years, biochemical, genetic and molecular biology studies have revealed that complex pathways are required in order that nascent hydrogenases mature into physiologically competent enzymes. This chapter covers the pathways which are required for the insertion of Fe, Ni and, in some enzymes, Se at the active site and for the transport of the enzyme into its correct physiological compartment. Some aspects of the maturation process, e.g. the formation and insertion of the bimetallic Ni-Fe site, are specific for hydrogenases but other aspects, e.g. the formation of [4Fe-4S] clusters and the membrane translocation pathway, are shared with other metalloproteins. This chapter does not address the biosynthesis of certain prosthetic groups, e.g. flavin adenine nucleotides (FAD, FMN), which though found in some hydrogenases are present in many other enzymes. Likewise there are many Fe-containing proteins in cells and Fe acquisition and metabolism has been reviewed extensively, and will only be mentioned herein. However, Ni is present in a few enzymes: hydrogenases, CO dehydrogenase and urease. Likewise Se occurs in relatively few enzymes and its insertion into proteins such as hydrogenases and formate dehydrogenase occurs via a remarkable co-translational mechanism. It is widely accepted that specific proteins known as chaperonins are required to control the folding of many proteins in vivo. There is much evidence to show that additional proteins, the so-called ‘accessory proteins’, are required to insert requisite metal centres in a manner which must be coordinated with the folding pathway. The complexity of these processes explains in part the usually high numbers of genes specifically required for hydrogenase activity. The complexity of the maturation processes for hydrogenases has implications for biotechnology. For example, to enhance expression of hydrogenase activity in any particular organism we must boost not only the production of the structural genes per se but may also need to enhance all those factors required for maturation.