P-glycoprotein (Pgp) overexpression has been shown to be correlated with resistance to chemotherapy in patients with malignant breast tumors. The aim of our study was to investigate the role of technetium-99m-tetrofosmin (99mTc-TF) as a functional imaging agent reflecting Pgp expression in these tumors. We prospectively studied 28 patients (26 females, 2 males; mean age, 53.07±9.88 years; range, 38 to 70 years) with breast cancer to ascertain the relationship between the degree of accumulation (lesion/nonlesion=L/NL) and percentage washout (WO%) rate of 99mTc-TF and expression of Pgp in tumor tissues. All patients received 555-740 MBq of 99mTc-TF intravenously at the arm controlateral to the suffering breast. Planar images were obtained 10 and 120 min post injection from prone lateral and anterior views with an acquisition time of 5 min. Visual and semiquantitative measurements were performed. The L/NL ratios and WO% rates were calculated semiquantitatively. Immunohistochemical studies were performed on paraffin sections using a monoclonal antibody, JSB-1. The L/NL ratios and WO% rates were related with the level of Pgp determined immunohistochemically. Our results showed an inverse correlation between the L/NL ratios of 99mTc-TF and the density of Pgp expression in tumor tissues, whereas there was no appreciable correlation between the tumor WO% rates of 99mTc-TF and the level of Pgp expression (P=0.275). The values for the L/NL ratios were significantly lower for those tumors expressing Pgp at high levels than those with intermediate or no Pgp expression (P<0.002 and P<0.04). In conclusion, although our results warrant further studies, our data strongly suggest that 99mTc-TF imaging is useful to noninvasively determine the presence of multidrug resistance in patients with breast cancer.