The crystal structure of Clostridium acidi-urici ferredoxin has been determined using multiple wavelength anomalous diffraction (MAD) techniques at 5.0-A resolution. The electron density map shows striking similarity to a map of Peptococcus aerogenes ferredoxin computed at the same resolution from the atomic coordinates reported by Adman et al. (Adman, E. T., Sieker, L. C., and Jensen, L. H. (1973) J. Biol. Chem. 248, 3987-3996). Such similarity is expected from the high degree of identity between amino acid sequences of the two proteins. The use of MAD methods has in the relatively recent past become a practical possibility due to instrumental advances enabling the collection of accurate data at several wavelengths at synchrotrons and due to theoretical and computational advances that facilitate the analysis of these data for the determination of phases. These methods hold great promise as an alternative to the multiple isomorphous replacement method in macromolecular structure determination. The present report represents one of the first applications of the MAD techniques to the determination of the structure of a protein which was previously unknown in detail.