We report the results of a Mössbauer study of the low-potential iron-sulfur cluster FX in the Photosystem I core protein of Synechococcus 6301. The Mössbauer spectrum of FX in the oxidized state shows an isomer shift of 0.42 mm/s, which is in good agreement with the 0.43 mm/s isomer shift found in [4Fe-4S] proteins but not with the isomer shift of 0.26 mm/s found in [2Fe-2S] proteins. In the reduced state the spectrum is asymmetrically broadened at 80 K, indicating the presence of two very closely spaced doublets with an average isomer shift of 0.55 mm/s, which is also in agreement with [4Fe-4S] proteins. At 4.2 K, the spectrum exhibits broadening and magnetic splitting similar to what is observed for [4Fe-4S] proteins and quite unlike [2Fe-2S] proteins. Given the assumption that the iron atoms of FX are tetrahedrally coordinated with sulfur ligands, the data strongly support the assignment of FX as a [4Fe-4S] cluster.