The aim of this study was to compare uptake of 99mTc-MIBI, 99mTc-tetrofosmin and 99mTc-Q12 in vitro and biodistribution in vivo in rats. In vitro, uptake decreased in the order MIBI-->tetrofosmin-->Q12. Uptake of MIBI and tetrofosmin, but not of Q12, in cultured tumor cells was dependent on the plasma membrane and mitochondrial potential. In vivo, heart uptake of all three compounds was high and stable. Tumor uptake decreased in the order MIBI-->Q12-->tetrofosmin and the tumor/blood ratio in the order MIBI-->tetrofosmin-->Q12.