The kinetics, intracellular distribution and effects of ion transport inhibitors on the uptake of 99Tcm-tetrofosmin, 99Tcm-methoxyisobutyl isonitrile (99Tcm-MIBI) and 201Tl were investigated in a tumour cell line. Both uptake and washout of the tracers were measured at specific intervals. Cells were treated with ouabain, dimethyl amiloride (DMA) and bumetanide to observe the effects on uptake, while carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used to study the release of the tracers. The tracers showed similar uptake and washout kinetics. Ouabain inhibited 55-67% of 201Tl, 16-22% of 99Tcm-tetrofosmin and 8-14% of 99Tcm-MIBI. DMA inhibited the uptake of both 99Tcm-tetrofosmin (30%) and 99Tcm-MIBI (21%). Bumetanide stimulated the uptake of 99Tcm-tetrofosmin and 99Tcm-MIBI but inhibited that of 201Tl (37%). When cells were pretreated with various combinations of these ion transport inhibitors, the uptake of 201Tl was related to Na+,K+ pump, Na+,K+, 2Cl- co-transport systems, whereas the uptake of both 99Tcm-tetrofosmin and 99Tcm-MIBI was related to the Na+,K+ pump, Na+/H+ antiport systems. Addition of CCCP released 55% and 90% of accumulated 99Tcm-tetrofosmin and 99Tcm-MIBI respectively, indicating that the percentage released was related to mitochondrial uptake.