A vanadium- and iron-containing cluster has been shown previously to accumulate on VnfX in the A. vinelandii mutant strain CA11.1 (deletion nifHDKvnfDGK::spc). In the present study, we show the homocitrate-dependent transfer of ⁴⁹V label from VnfX to nif-apodinitrogenase in vitro. This transfer of radiolabel correlates with acquisition of acetylene reduction activity. Acetylene is reduced both to ethylene and ethane by the hybrid holodinitrogenase so formed, a feature characteristic of alternative nitrogenases. Structural analogues of homocitrate prevent the acetylene reduction ability of the resulting dinitrogenase. Addition of NifB-co or a source of vanadium (Na₃VO₄ or VCl₃) does not increase nitrogenase activity. Our results suggest that there is in vitro incorporation of homocitrate into the V-Fe-S cluster associated with VnfX and that the completed cluster can be inserted into nif-apodinitrogenase. The homocitrate incorporation reaction and the insertion of the cluster into nif-apodinitrogenase (₂₂₂) do not require MgATP. Attempts to achieve FeV-co synthesis using extracts of other FeV-co negative mutants were unsuccessful, showing that earlier steps in FeV-co synthesis, such as the steps requiring VnfNE or VnfH, do not occur in vitro.