Neutral Re(V) and Tc(V) oxo complexes of the peptide dimethylglycyl-L-seryl-L-cysteinylglycinamide (RP294) were prepared and characterized by HPLC, spectroscopic techniques, and X-ray crystallographic analysis. The peptide was prepared as a single peptide chain using solid phase methods and characterized by HPLC and various spectroscopic techniques. The water-soluble Re(V) oxo complex of dimethylglycyl-L-seryl-L-cysteinylglycin [ReO(RP294)] was prepared from the reaction of the peptide with either [ReO2(en)(2)]CL or ReOCl3(PPh3)(2) in the presence of base. The complex exists as two isomers, the serine CH2OH group being in the syn or anti conformation with respect to the Re-oxo bond. The ratio of the isomers at room temperature is 1:1.1. The isomers were separated by reverse-phase HPLC, but the isolation of each isomer was complicated by their rapid interconversion in aqueous solution at room temperature. The molecular structure of the syn isomer of the Re complex was determined by X-ray crystallography. Crystals of syn-[ReO(RP294)] (C12H20N6O5ReS) are orthorhombic, of space group P2(1)2(1)2(1), with a = 6.954(1) Angstrom, b = 8.0472(1) Angstrom, c = 32.9183(4) Angstrom, and Z = 4. The structure was solved by direct methods and was refined by full-matrix least-squares procedures to R = 0.0327 (R-w = 0.0838) for 10447 reflections with I > 2 sigma(I). The Re metal was coordinated in a distorted square pyramidal geometry with the oxo moiety in the apical position. The peptide coordinated to ReO3+ via the N-amine atom of dimethylglycine, the S-thiolate atom of cysteine, and the two N-amide atoms of serine and cysteine (an N2N'S donor atom set). The Re atom lies similar to 0.74 Angstrom above the distorted plane formed by the N2N'S donor atom set. Variable-pH H-1 NMR spectral data showed the Re complex was stable from pH 5 to 8.5. The reaction of (TcO4-)-Tc-99 with SnCl2, sodium gluconate, and RP294 produced the Tc-99(V)oxo RP294 complex, [(TcO)-Tc-99(RP294)]. Like the [ReO(RP294)] complex, [(TcO)-Tc-99(RP294)] also exists in the syn and anti conformations in a ratio of approximately 1:1. The Tc-99m complex of RP294 was prepared at the tracer level from the reaction of Na[(TcO4)-Tc-99m] with excess SnCl2, sodium gluconate, and RP294. The Tc-99m and Re RP294 complexes behaved similarly under identical HPLC conditions.