Multi-drug resistance mediated by the transmembrane pump P-glycoprotein (Pgp) is an important mechanism of resistance of certain tumours against chemotherapeutic drugs. The myocardial perfusion imaging agent 99Tcm-sestamibi is a substrate for Pgp. Further characterization of 99Tcm-sestamibi has now been carried out in the transplantable rat breast adenocarcinoma cell line, MatB, and its doxorubicin-resistant variant, AdrR. In vitro accumulation of the tracer in wild-type (WT) MatB was high and was not affected by the Pgp modulator, PSC833. Conversely, AdrR cells did not accumulate significant amounts of tracer unless PSC833 was present. Imaging studies in rats bearing MatB-WT and AdrR tumours showed that 99Tcm-sestamibi washed out of the resistant tumours at three times the rate of WT tumours. These results support the potential use of 99Tcm-sestamibi for functional imaging of Pgp activity in patients undergoing chemotherapy.