Pretargeting with bivalent effectors capable of bridging antitumor antibodies (affinity enhancement pretargeting) has been reported to provide superior results by affinity enhancement. Phosphorodiamidate morpholinos (MORFs) and other DNA analogues used for pretargeting are ideally suited as bivalent effectors since they are easily synthesized and the distance between binding regions, a determinant of binding, may be adjusted simply by lengthening the chain. We have shown by surface plasmon resonance that bivalent MORFs will provide superior affinity enhancement provided that suitable spacing exists between the binding regions. The goals of this study were to synthesize a bivalent MORF with a MAG(3) group attached for technetium-99m ((99m)Tc) radiolabeling, investigate whether the bivalent MORF showed improved cell accumulation in culture compared to its corresponding monovalent MORF and compare biodistributions in normal mice and in pretargeted tumored mice. An excess of an amine derivatized 18 mer MORF with 6 nonbinding bases for spacing was reacted with Fmoc-l-beta-homoglutamic acid to form duplexes via their carboxylate groups and, after deprotection, conjugated with NHS-MAG(3) to attach the chelator. The anti-CEA antibody MN14 was conjugated with a 12 mer complementary MORF (i.e., cMORF). The binding behavior between radiolabeled monovalent and bivalent MORFs was compared in LS174T tumor cells at 4 degrees C pretargeted with MN14-cMORF. Biodistributions of radiolabeled monovalent and bivalent MORFs at 3 h postadministration were measured in normal mice and in tumor mice pretargeted with MN14-cMORF. In the pretargeted cells in culture, the accumulation of the bivalent MORF was significantly higher than the monovalent MORF (p = 0.002), thus providing strong evidence for affinity enhancement. In normal mice, whole body clearance of the bivalent and monovalent MORFs was equally rapid. In tumored mice, tumor accumulation of the radiolabeled bivalent MORF was significantly higher than that of the monovalent MORF. In conclusion, a bivalent MAG(3)-MORF was successfully synthesized and radiolabeled with (99m)Tc. While a pharmacokinetic effect for the higher tumor accumulations in pretargeted mice of the radiolabeled bivalent MORF cannot be excluded, the results may be best explained by affinity enhancement. Thus two monovalent MORFs were covalently conjugated into a bivalent MORF effector to improve tumor targeting by both pharmacokinetics and affinity enhancement influences.