The coordination sphere of Mn(II) in the complex with GDP and elongation factor Tu from Escherichia coli has been probed by EPR spectroscopy with 17O-labeled ligands. Inhomogeneous broadening in the EPR signals for Mn(II) due to unresolved superhyperfine coupling to the 17O nucleus was used to identify directly bound oxygen ligands. Results with GDP selectively enriched with 17O either in the alpha-phosphate or in the beta-phosphate revealed that GDP was a beta-monodentate ligand for Mn(II) in the complex with the protein. Results with 17O-enriched water showed that two water molecules are coordinated to the Mn(II). The EPR spectrum for the complex is characteristic of octahedral coordination for Mn(II). Hence, three ligands from the protein are required to complete the sextet of ligands.