99mTc-sestamibi (99mTc-MIBI) is a substrate for the P-glycoprotein (P-gp) pump but it is not known whether it is a substrate for the multidrug resistance-associated protein (MRP) pump. Therefore, 99mTc-MIBI was evaluated in the GLC4 cell line and its doxorubicin-resistant MRP-, but not P-gp-, overexpressing GLC4/ADR sublines as well as in the S1 cell line and its MRP-transfected subline S1-MRP. 99mTC-MIBI concentration decreased in the GLC4/ADR sublines with increasing MRP overexpression and was lower in S1-MRP than in S1. 99mTC-MIBI plus vincristine increased 99mTc-MIBI concentration in GLC4 lines compared with 99mTc-MIBI alone. 99mTc-MIBI efflux raised with increasing MRP expression in the GLC4 lines. Glutathione depletion elevated 99mTC-MIBI concentration in GLC4/ADR(150x). Cross resistance for 99Tc-MIBI, used to test cytotoxicity of the Tc compound, was observed in GLC4/ADR(150x) vs GLC4. 99Tc-MIBI induced a synergistic effect on vincristine cytotoxicity in GLC4/ADR(150x). These results show that 99mTc-MIBI is involved in MRP-mediated efflux. The fact that 99mTc-MIBI efflux is influenced by MDR1 and MRP expression must be taken into account when this γ-rays-emitting complex is tested for tumour efflux measurements.