A survey of ferredoxin maquettes derived from natural sequences was utilized to obtain a primary sequence competent for [4Fe-4S](2+/+) incorporation for the study of the minimal ligand requirements for cluster assembly. The resultant 16 amino acid ferredoxin maquette (FdM), NH2-KLCEGG . CIACGAC . GGW-CONH2, incorporates a single [4Fe-4S](2+/+) cluster as evidenced by a cluster titration assayed by electron paramagnetic resonance (EPR) spectroscopy. Assembly of the [4Fe-4S] cluster within FdM was kinetically facile (minutes time scale) and the cluster was stable in solution under strictly anaerobic conditions. The four cysteines of FdM were systematically replaced with nonligating alanine residues resulting in lower yields (10-56% relative to a FdM control) as quantitiated in the reduced state by EPR spectroscopy demonstrating the necessity for each in successful cluster incorporation. A single example of a cysteine to leucine modification resulted in a lower yield of cluster incorporation equivalent to the analogous alanine replacement indicating a general absence of steric hindrance for [4Fe-4S] assembly in these small peptides. Pairwise replacement of cysteines with alanines resulted in dramatic loss of yield and cofactor induced assembly of a pair of peptides as evidenced by EPR spectroscopy and size exclusion chromatography, respectively. Alanine substitution of three of the four cysteines from the FdM sequence resulted in a virtual loss of [4Fe-4S] incorporation ability. Attempts were made to provide non-cysteine ligands to the incorporated cluster using aspartic acid and histidine residues; however, the lower yield of [4Fe-4S] assembly in these peptides coupled with both the identical EPR spectral parameters and redox potential indicates a lack of observable aspartate or histidine ligation to the clusters. The measured yields of [4Fe-4S] incorporation provide a convenient tool for probing the basic ligand requirements for the [4Fe-4S](2+/+) cluster in these 16 amino acid ferredoxin maquettes.