We have examined the iron-sulfur cluster of aconitase, a high-potential iron-sulfur protein, by absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectroscopy. The MCD spectrum of unactivated aconitase, which is presumably oxidized, is similar to those of reduced two iron-two sulfide ferredoxins but distinct from the MCD of known four iron-four sulfide proteins. The magnitude of the natural CD of unactivated aconitase also suggests the absence of four iron-four sulfur clusters. Reduction of the enzyme with dithionite and activation with the cysteine-ascorbate-ferrous ion activation mixture generate spectra which are significantly different from those of any iron-sulfur protein seen to date. We interpret these results as indicating that aconitase does not contain a four iron-four sulfur cluster generally thought to be characteristic of high-potential iron-sulfur proteins. It could contain a two iron-two sulfur center or some other center such as a cyclic three iron-three sulfur center.