This laboratory is evaluating phosphorothioate deoxyribonucleic acids (DNAs) and peptide nucleic acids (PNAs) for a variety of nuclear medicine applications. Morpholinos (MORFs) are a new class of oligomers with a nuclease-resistant, nonionic and water-soluble phosphorodiamidate backbone. We now report on the in vitro and in vivo properties of MORFs labeled with technetium-99m. Both 15-mer and 18-mer MORFs were obtained, each with a primary amine attached to the 3' equivalent end via a three-carbon beta-alanine linker. The amine was used to conjugate with NHS-MAG3 for 99mTc radiolabeling. By surface plasmon resonance at room temperature, the association rate constant for hybridization of the 18-mer MORF to its complementary oligomer (cMORF) was equivalent to that of DNAs and PNAs of comparable length. Hybridization of 99mTc-MORF in vitro to free cMORF, to a cMORF polymer and to cMORF beads was nearly quantitative under a variety of conditions. Kinetic studies in vitro at room temperature showed rapid (2-5 min) and nearly quantitative (90%) binding to cMORF beads. Using size-exclusion high-performance liquid chromatography, the stability of the 99mTc-MORF was found to be greater than 85% over 24 h in 37 degrees C serum with minimal protein binding. In normal mice, the 99mTc-MORF showed rapid pharmacokinetics, with only 21% and 8% remaining in the whole body at 3 and 24 h post administration, respectively. In vivo targeting with 99mTc-MORF of cMORF beads in one thigh of normal mice compared to control beads in the other thigh showed target/control thigh ratios of 2-10 between 3 and 24 h. These results demonstrate that MORF oligomers are capable of in vivo hybridization. Their properties of hybridization affinity and kinetics and their in vivo stability and pharmacokinetics make them suitable subjects for in vivo studies.