We investigated the kinetics, cellular uptake and intracellular distribution of 99mTc-tetrofosmin in tumor cell lines and compared them with those of 99mTc-MIBI and 201TI. METHODS At specific intervals after incubation with radiotracers, cellular uptake was determined. Cells were also treated with nigericin, carbonyl cyanide m-chloro-phenylhydrazone (CCCP) and ouabain to determine their effects on the uptake of the tracers. RESULTS Each tracer showed similar uptake kinetics in both cell lines, and a steady-state was maintained for at least 4 hr. Nigericin stimulated the uptake of both 99mTc-tetrofosmin and 99mTc-MIBI in HBL-2 cells, although it inhibited their uptake in SW-13 cells. Nigericin also inhibited 90% of 201TI uptake in both cell lines. Addition of CCCP caused 73%-97% release of accumulated 99mTc-MIBI from both cell lines with or without nigericin pretreatment, indicating that most of the accumulated 99mTc-MIBI was related to mitochondria. The effect of CCCP on accumulated 99mTc-tetrofosmin was less marked than that on 99mTc-MIBI in both cell lines, indicating that only a part of accumulated 99mTc-tetrofosmin, was related to mitochondria. Ouabain preincubation inhibited 74%-77% and 51%-53% of 201TI uptake in HBL-2 and SW-13 cells, respectively, as well as inhibited 22%-31% uptake of 99mTc-tetrofosmin in both HBL-2 and SW-13 cells. Uptake by the dead cells of either cell line was negligible for each tracer. CONCLUSIONS Technetium-99m-tetrofosmin uptake depends on both cell membrane and mitochondrial potentials. Only a small fraction of 99mTc-tetrofosmin accumulates inside the mitochondria, while most 99mTc-MIBI accumulates inside the mitochondria. Thallium-201 uptake is partly independent of the Na+, K+ pump.