Introduction: When a radiopharmaceutical is simultaneously administered with a medicine that has high affinity for the same plasma protein, the radiopharmaceutical is released at higher concentrations in blood, leading to enhanced transfer into target tissues. This is known as the serum protein binding displacement method. In this study, we investigated the pharmacokinetic alteration of technetium-99m-labeled mercaptoacetylglycylglycylglycine (99mTc-MAG3) using the serum protein binding displacement method. Methods: Rat and human serum protein binding rates of 99mTc-MAG3 were measured by ultrafiltration with or without displacers of human serum albumin (HSA) binding sites I and II (200μM and 400μM loading). Male Wistar rats were injected with 99mTc-MAG3 (740kBq/0.3mL saline) via the tail vein, and biodistribution was assessed at 2, 5, 10 and 15min. Dynamic whole-body images were obtained for 99mTc-MAG3 (11.1MBq/0.3mL saline)-injected rats, with or without HSA displacers. Results: 99mTc-MAG3 strongly bound to HSA (87.37%±2.13%). Using HSA site I displacers, the free fraction of 99mTc-MAG3 increased significantly (1.20 to 1.47 times) when compared with controls. For biodistribution and imaging, rapid blood clearance was observed with bucolome (BCL) loading, which is an HSA site I displacer. With BCL loading, peak times for rat renograms were respectively shifted from 240s to 110s, and from 170s to 120s. Conclusions: We found that 99mTc-MAG3 bound to the HSA binding site I. It was confirmed that pharmacokinetic distribution of 99mTc-MAG3 is altered by presence of BCL, which leads to increases in the free fraction of 99mTc-MAG3, and BCL produced rapid blood clearance and fast peak times on rat renograms. The serum protein binding displacement method using 99mTc-MAG3 and BCL, a safe displacer for humans, may be applicable to clinical study and lead to better diagnostic images with shorter waiting times and lower radiation doses for patients. © 2013 Elsevier Inc.