Burkholderia cepacia AC1100 is able to use the chlorinated compound 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as the sole source of carbon and energy. CW EPR and one-dimensional ESEEM spectroscopy studies performed earlier indicate the presence of a Rieske-type [2Fe-2S] cluster with two coordinated histidine residues in 2,4,5-T monooxygenase from B. cepacia. This paper describes the application of two-dimensional ESEEM (called HYSCORE) spectroscopy for further characterization of the nitrogens surrounding the reduced Rieske-type cluster. The HYSCORE spectra measured at field positions in the neighborhood of the principal directions of the g tensor contain major contributions from cross-peaks correlating the two double-quantum transitions from each histidine nitrogen. These allow the estimation of the diagonal components of the hyperfine tensors along the principal axes of the g tensor : 4.05, 3.88, and 4.01 MHz (N1) and 4.71, 5.07, and 5.02 MHz (N2). Other spectral features from the histidine nitrogens usually have a much weaker intensity and are occasionally observed in the spectra. HYSCORE measurements have been also performed with the reduced [2Fe-2S] plant ferredoxin-type cluster with four cysteine ligands in a ferredoxin from Porphira umbilicalis, and spectral features produced by the peptide nitrogen are observed. Similar features also appear in the HYSCORE spectra of the Rieske cluster. Systematic differences are observed between 2,4,5-T monooxygenase and published results from related benzene and phthalate dioxygenases that may reflect structural and functional differences in histidine ligation and the nitrogens of nearby amino acids in Rieske-type [2Fe-2S] clusters.