Chemotherapeutic treatment of cancer patients is often unsuccessful, due to the involvement of various mechanisms, leading to multidrug resistance (MDR). In this review, I describe the mechanisms involved in MDR. Furthermore, results obtained by imaging of P-glycoprotein (P-gp) and the multidrug resistance associated protein (MRP) are reviewed. Single photon emission computed tomography (SPECT) and positron emission tomography (PET) are unique techniques to study P-gp- and MRP-mediated transport. The radiopharmaceutical 99mTc-sestamibi is a substrate for both P-gp and MRP. This tracer has been used for tumor imaging in clinical studies, and to visualize blockade of P-gp mediated transport after modulation of the P-gp pump. Other 99mTc-radiopharmaceuticals such as 99mTc- tetrofosmin and several 99mTc-Q-complexes are also substrates for P-gp. Until now, for these compounds only results from in vitro and animal studies are available. For quantification of P-gp mediated transport with PET in vivo, several agents, such as [11C]colchicine, [11C]verapamil and [11C]daunorubicin have been evaluated. In vivo results suggest that these radiopharmaceuticals can be used to image P-gp function in tumors. 124I and 76Br radiolabeled doxorubicin analogues are also useful to examine P-gp mediated transport. Leukotrienes are specific substrates for MRP. Therefore, N-[11C]acetyl-leukotriene E4 provides the opportunity to study MRP function non-invasively. Results obtained with this radiopharmaceutical in MRP2 mutated GY/TR- rats indicate visualization of MRP-mediated transport. This tracer enables to study MRP transport function abnormalities in vivo such as in Dubin-Johnson patients, who are MRP2 gene deficient. In conclusion, it is feasible to study the functionality of MDR transporters in vivo, both with SPECT and with PET. Such imaging techniques may become an important factor in the development of novel chemotherapeutic drugs.