The biosynthesis of the [NiFe]- and [FeFe]-hydrogenase enzymes requires the activities of multiple proteins that assemble the intricate metallocenters on the enzyme precursor proteins in an energy-dependent process. These accessory proteins include enzymes that synthesize the non-protein iron ligands as well as metallochaperones for the delivery of nickel to the [NiFe]-hydrogenase. Over the past few years many of these proteins have been examined in vitro. The biochemical properties, in the context of the earlier genetic studies, provide a basis for assigning function to the individual accessory proteins and mapping out the sequential steps of the metallocenter assembly pathways. This framework will serve as a foundation for detailed mechanistic analysis of these complex biomolecular factories.