HydE, HydF, and HydG participate in the synthesis of the complex di-iron center of [FeFe] hydrogenases. The hydE, hydF, hydG, hydA, and hydB genes of Desulfovibrio vulgaris Hildenborough were cloned and His-tag pull-down assays were used to study the potential interaction between HydE, HydF, and HydG with the HydA and HydB protein subunits of the D. vulgaris [FeFe] hydrogenase. Interaction of HydE and HydG with HydA was demonstrated. HydF did not interact with HydA, and none of the accessory proteins appeared to interact with HydB. This suggests that specific protein-protein interactions may be required during [FeFe] cluster synthesis and/or insertion.