Since 201Tl, 99mTc-sestamibi and 99mTc-teboroxime concentrate in cardiac cells through different mechanisms, we compared their uptake in cultured normal cells and carcinoma cell lines in order to define their possible use for tumor evaluation in vivo. Four lines of normal cells from animals, including myocytes from newborn rats, and four lines of human carcinoma cell lines were incubated for 1 hr with 37 kBq of either tracer. Results, expressed in percent of the total activity taken up by 1 million cells, showed a 9% difference between the uptake of teboroxime by normal and carcinoma lines (24.6% +/- 2.8% versus 22.5% +/- 2.1%, respectively, p < 0.05). Mean uptake was 80% higher in tumor than in normal cells for 201Tl (5.39% +/- 1.33% versus 3.00% +/- 1.08%, respectively, p < 0.001) and nearly 4 times higher for sestamibi (5.37% +/- 2.34% versus 1.44% +/- 1.88%, p < 0.001). For both agents, uptake by the myocytes and carcinoma cells was comparable (5.14% +/- 0.11% for 201Tl and 5.28% +/- 1.03% for sestamibi). When the myocytes are excluded from the group of normal cells, the uptake is 112% higher in tumor than in normal cells for 201Tl (5.39% +/- 1.33% versus 2.54% +/- 0.44%, p < 0.001) but it becomes nearly nine times higher for sestamibi (5.37% +/- 2.34% versus 0.60% +/- 0.23%, p < 0.001). It is concluded that these experiments show that the uptake of sestamibi was the most discriminant to separate between normal and malignant cells, while teboroxime was the less discriminant. Potential clinical applications for tumor visualization based on differences in sestamibi and teboroxime uptake could be envisioned.