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Azotobacter vinelandii has recently been used for a variety of genetic experiments which take advantage of its facile transformation system and its high-frequency homologous recombination. One gene that has been cloned and sequenced is the fdxA gene that encodes a small Fe-S protein called A. vinelandii ferredoxin I (AvFdI). Because this protein has been extensively characterized by X-ray crystallography and spectroscopic methods, we are using it as a model to address some general questions in Fe-S biochemistry. AvFdI is not a very abundant protein in wild-type cells, so to facilitate our biochemical studies we have developed the overexpression system described herein. The results show that AvFdI can be easily overproduced ca. 50-fold in its native background, by introducing multiple copies of the fdxA gene into A. vinelandii, on the broad-host-range multicopy plasmid, pKT230. The protein can be expressed from its own constitutive promoter or from the controlled nifH promoter. The overproduced protein has no deleterious effects on the organism and is identical to the protein produced by wild-type cells. This A. vinelandii-based system should be generally useful for the overproduction of other A vinelandii proteins or for the expression of genes from thermophilic or other organisms with similarly high G-C contents, or for the expression of O2-sensitive metalloproteins that are unstable in other systems.