Technetium-99m hexakis-2-methoxyisobutylisonitrile (MIBI), 99mTc-tetrofosmin and 99mTc-Q12 were all introduced for myocardial imaging but found additional applications as they are taken up by different tumours, enabling imaging of these lesions in patients. The aim of this study was to compare the uptake characteristics of these compounds in vitro in the human adenocarcinoma breast cell lines MCF-7 and ZR-75. It was shown that 99mTc-MIBI had the highest cellular uptake (15.9%+/-0.5% dose/mg protein after 60 min in MCF-7, and 14.2%+/-0.4% dose/mg protein in ZR-75), followed by 99mTc-tetrofosmin (6.8%+/-0.6% dose/mg protein in MCF-7, and 8. 2%+/-0.2% dose/mg protein in ZR-75) and 99mTc-Q12 (3.2%+/-0.1% dose/mg protein in MCF-7, and 3.5%+/-0.3% dose/mg protein in ZR-75 cells). For all three compounds tenfold differences in specific activity did not influence total cell-associated radioactivity. Uptake of 99mTc-MIBI and 99mTc-tetrofosmin was obviously lower at 4 degrees C than at 37 degrees C, whereas 99mTc-Q12 uptake showed only slight temperature dependence. When uptake was compared in cells grown to different cell densities (1 mg/ml cellular protein versus 0.3 mg/ml), no differences in uptake were detected when uptake was corrected for the amount of cellular protein present in the dishes. Furthermore, for all compounds it was shown that cellular radioactivity decreased rapidly after washing. Apart from the differences in cellular uptake of the three compounds after 60 min, no differences in residual cellular radioactivity after washing were found between the different compounds when expressed as a percentage of their 60-min uptake, suggesting that the efflux process of the radiolabelled compounds was similar. The differences in cell-associated activity after 60 min were thus presumably caused by differences in uptake. It was concluded that of the Tc-labelled compounds tested, 99mTc-MIBI had the highest cellular retention in both human breast tumour cell lines. However, for imaging in vivo not only radioactivity in the target organ is important, but also the ratio of radioactivity in the target versus that in the background. Therefore, further studies in vivo need to be performed to investigate which compound is the optimal imaging agent.