We conjugated mercaptoacetyltriglycine (MAG(3)) to rh-annexin V to permit radiolabeling with (99m)Tc in an effort to decrease the high kidney and liver accumulation observed for (99m)Tc-labeled Hynic-annexin V. The 36-kDa protein was conjugated at a 5:1 molar ratio with NHS-MAG(3) in HEPES buffer pH 7.8 at room temperature, then quenched with glycine and purified by dialysis. The biopotency of the resulting MAG(3)-annexin was similar to that of Hynic-annexin as determined by a sensitive red blood cell membrane affinity binding assay and a surface plasmon resonance (SPR) assay. The (99m)Tc radiolabeling of MAG(3)-annexin resulted in radiochemical yields of 90% under mildly basic pH conditions. Biodistribution data in normal mice clearly showed a significant decrease in kidney and liver uptake at 1 h postinjection for the (99m)Tc MAG(3)-annexin compared to the (99m)Tc Hynic-annexin (from 24% ID to 4% ID for the liver, and 45% ID to 15% ID for the kidneys, respectively). Autoradiography of the kidneys showed retention of radioactivity in the collecting tubules following administration of both labeled annexins. The (99m)Tc MAG(3)-annexin biodistribution was also characterized by a lower retention of radioactivity in the whole body, but with small intestine accumulation over fivefold higher than observed with (99m)Tc Hynic-annexin. These findings show a definite improvement in renal and hepatic clearance of the MAG(3) radioligand. However, due to the increased radioactivity uptake in the small intestines, the early in vivo detection of ongoing apoptosis in the lower abdomen might be more difficult with (99m)Tc MAG(3)-annexin. Nevertheless, (99m)Tc MAG(3)-annexin may be an attractive alternative to (99m)Tc Hynic-annexin for the in vivo imaging of phosphatidylserine receptors.