We investigated the renal clearance, the extrarenal clearance, the biodistribution, the plasma extraction ratio and the imaging quality on a scintillation camera of a new substance: 99mTc-mercaptoacetyltriglycine (MAG-3) in rats. Simultaneously 125I-hippurate (OIH) and 51Cr-EDTA were used as reference substances. In scintillation camera studies, 123I-hippurate served as reference. MAG-3 was prepared by kit labelling without further purification, as it would be used in clinical studies. However in three separate rats, HPLC purified MAG-3 was used. High renal clearance and extraction ratio was found but these were less than that for OIH (1.95 ml/min per 100 g BW vs 2.76 ml/min per 100 g BW and 64% vs 85% respectively). The extrarenal clearance was higher for MAG-3 than for OIH (0.20 ml/min per 100 g BW vs 0.04 ml/min per 100 g BW), presumably because of bile excretion. HPLC purification increased the renal excretion of MAG-3: the clearance was 2.53 ml/min per 100 g BW, the extraction fraction 75%, but the extrarenal clearance was unchanged. The imaging quality was comparable to that of 123I-hippurate in early pictures, but extrarenal activity, presumably representing bile, was observed in late pictures. We conclude that MAG-3 has some potential for the replacement of OIH but clinical studies are necessary to test whether the same conditions and limitations exist in humans.