The reaction of a class I ribonucleotide reductase (RNR) begins when a cofactor in the beta subunit oxidizes a cysteine residue similar to 35 angstrom away in the alpha subunit, generating a thiyl radical. In the class lc enzyme from Chlamydia trachomatis (Ct), the c-ysteine oxidant is the Mn-IV ion of a Mn-IV/Fe-III cluster, which assembles in a reaction between O-2 and the Mn-II/Fe-II complex of beta. The heterodinuclear nature of the cofactor raises the question of which site, 1 or 2, contains the Mn-IV ion. Because site 1 is closer to the conserved location of the cysteine-oxidizing tyrosyl radical of class Ia and Ib RNRs, we suggested that the Mn-IV ion most likely resides in this site (i.e., Mn-I(IV)/Fe-2(III)) out a subsequent computational study favored its occupation of site 2 (F-I(III)/Mn-2(IV)). In this work, we have sought to resolve the location of the Mn-IV ion in Ct RNR-beta by correlating X-ray crystallographic anomalous scattering intensities with catalytic activity for samples of the protein reconstituted in vitro by two different procedures. In samples containing primarily (MnFeIII)-Fe-IV/ clusters, Mn preferentially occupies site 1, but some anomalous scattering from site 2 is observed, implying that both and Mn-I(II)/Fe-2(II) and Fe-I(II)/Mn-2(II) complexes are competent to react with O-2 to produce the corresponding oxidized states. However, with diminished Mn-II loading in the reconstitution, there is no evidence for Mn occupancy of site 2, and the greater activity of these "low-Mn" samples on a per-Mn basis implies that the Mn-I(IV)/Fe-2(III)-beta is at least the more active of the two oxidized forms and may be the only active form.