After administration of the 99mTc complex with N, N'-1,2-ethylenediylbis-L-cysteine diethyl ester (99mTc-ECD), a brain perfusion imaging agent, the radioactive metabolite is trapped in primate brain, but not in mouse and rat. Here, we investigate the involvement of metabolite extrusion by organic anion transporter 3 (OAT3), which is highly expressed at the blood–brain barrier in mice, in this species difference. The efflux rate of radioactivity in the cerebrum of Oat3−/− mice at later phase was 20% of that of control mice. Thus, organic anion transporters in mouse brain would be involved in the low brain retention of radioactivity after 99mTc-ECD administration.