We have recently shown that accumulation in mouse kidneys of technetium-99m labeled phosphorodiamidate morpholinos (MORFs) increases with the number of cytosines in the base sequence. To improve tumor/kidney ratios in tumored mice, pretargeting studies were performed with a cytosine-free MORF. An 18-mer MORF (5'-TCTTCTACTTCACAACTA) was conjugated to the anti-CEA antibody MN14 (Immunomedics) and administered to nude mice bearing LS174T tumors. Thereafter, the (99m)Tc-labeled cytosine-free cMORF (5'-TAGTTGTGAAGTAGAAGA-amide-MAG(3)) was administered. For comparison, the identical study was repeated but with our original pair of 18-mer MORFs (5'-GGGTGTACGTCACAACTA-conjugated MN14 and (99m)Tc-labeled 5'-TAGTTGTGACGTACACCC-amide-MAG(3)). Surface plasmon resonance was used to show that the hybridization affinities of the original and the modified pair of MORFs were essentially equal. Hybridization of the cytosine-free cMORF-(99m)Tc to MN14-MORF was demonstrated in vitro by size-exclusion high-performance liquid chromatography. At 3 h, kidney levels in normal mice were 2.0%ID/organ for the modified cMORF vs. 4.1%ID/organ for the original cMORF sequence, while at 24 h, these values were 0.9% vs 1.8%ID/organ. Pretargeting studies in tumored mice receiving 25 microg of conjugated antibody, 0.5 microg of labeled cMORF 48 h later, followed by imaging and sacrifice at 3 h showed that kidney levels were reduced using the cytosine-free cMORF. Moreover, tumor accumulation was about 3.6%ID/g and was independent of sequence. The whole-body images clearly reflected the improved tumor to kidney ratios. By choosing a cytosine-free base sequence for pretargeting studies, kidney accumulation of cMORF-(99m)Tc was reduced without adversely influencing tumor accumulation. The lowering of kidney radioactivity levels in this way may be important to reduce toxicity to this organ in connection with pretargeting radiotherapy studies.