The class I ribonucleotide reductase from Chlamydia trachomatis uses a stable Mn-IV/Fe-III cofactor to initiate nucleotide reduction by a free-radical mechanism. The enzyme provides the first example both of a Mn-dependent ribonucleotide reductase and of a Mn/Fe redox cofactor. In this work, we have used variable-field Mossbauer spectroscopy to demonstrate that the active cofactor has an S = 1 ground state due to antiferromagnetic coupling between the Mn-IV (S-Mn = 3/2) and high-spin Fe-III (S-Fe = 5/2) sites.