In vitro studies have shown that technetium-99m tetrofosmin (Tc-99m TF) is a transport substrate for the P-glycoprotein (Pgp) pump. Therefore, Tc-99m TF uptake of tumors can be used to predict chemotherapy response in lung cancers. However, whether lung resistance-related protein (LRP) expression affects tumor accumulation and efflux of Tc-99m TF in lung cancers is not known. Our aim was to use Tc-99m TF uptake of tumors to predict paclitaxel-based chemotherapy response of non-small cell lung cancer (NSCLC) and to compare Pgp or LRP expression. Twenty patients with advanced NSCLC received Tc-99m TF chest images before Taxol-based chemotherapy was used in this study. The chemotherapy response was evaluated by clinical and radiological methods in the third month after completion of treatment. No significant differences of prognostic factors (age, sex, body weight loss, performance status, tumor size, tumor stage, and tumor cell type) were found between the patients with good and those with poor responses. Early and delayed tumor/normal lung (T/L) uptake ratios were calculated on Tc-99m TF chest images. Immunohistochemical analyses were performed on multiple nonconsecutive sections of the biopsy specimens to detect Pgp and LPR expressions. The early and delayed T/L uptake ratios of 10 patients with good response were significantly higher than those of the other 10 patients with poor response. Significantly higher early and delayed T/L uptake ratios were found in patients with negative than those with positive Pgp expression ( p < 0.05). However, no significant differences of early and delayed T/L uptake ratios were found between patients with negative and positive LRP expressions ( p > 0.05). We found that Tc-99m TF imaging could accurately predict Taxol-base chemotherapy response. In addition, the Tc-99m TF tumor uptake was related to Pgp but not LPR expression in NSCLC.