Two radiopharmaceuticals, Tc-99m-MIBI (MIBI) and Tc-99m-Tetrofosmin (Tfos), are currently used for in vivo non-invasive monitoring of the MultiDrug Resistant (MDR) status of tumours. As gliomas are highly multidrug resistant, it is expected that the tracers would be poorly retained in those cells, but the in vivo and in vitro studies to date have shown that Tfos was highly retained in malignant gliomas. The high degree of malignancy of tumour cells is linked to alterations of physiological parameters as plasma membrane potential and intracellular pH. In order to elucidate the contribution of those parameters to Tfos and MIBI uptakes in malignant gliomas, we used several glioma cell lines--G111, G5, G152, and 42 MG-BA. These cells showed to be chemoresistant with a high level of expression and activity of the Multidrug Resistant associated Protein 1 (MRP1). They also had an alkaline intracellular pH (pHi) related to the Na+/H+ antiporter (NHE-1) expression and depolarised plasma membranes (-45 to -55 mV). In spite of their chemoresistance, we have found a high accumulation of both radiotracers in gliomas, more important for Tfos than MIBI, related to the presence and activity of NHE-1. In conjunction, the uptakes of the tracers were only partially dependent upon the plasma membrane potential of the glioma cell lines, again Tfos uptake being less dependent on this parameter than MIBI uptake. In conclusion, the evidence accumulated in this study suggests that Tfos could be a suitable glioma marker in vivo.