Objective: The aim of this study was to compare technetium-99m methoxyisobutylisonitrile (Tc-MIBI) chest single photon emission computed tomography (SPECT) results, immunohistochemical analyses of P-glycoprotein (Pgp) expression and response to paclitaxel in non-small-cell lung cancer (NSCLC). Methods: Before chemotherapy with paclitaxel, 30 patients with stage IIIb or IV NSCLC were enrolled in this study. Early chest SPECT was performed 10 min after intravenous injection of Tc-MIBI. Tc-MIBI chest SPECT images were qualitatively interpreted. Early tumor-to-normal lung ratios (T/NL) were calculated quantitatively. Immunohistochemical analyses were performed on multiple nonconsecutive sections of the biopsy specimens to determine Pgp expression. Chemotherapy response was evaluated in the third month after completion of treatment by clinical and radiological methods. Results: All 15 (100%) cases with good response and negative Pgp expression had positive results of early Tc-MIBI chest SPECT. Ten of 15 (67%) cases with poor response and positive Pgp expression had negative results of early Tc-MIBI chest SPECT. These early T/NL ratios (3.3 ± 0.8 for the 15 patients with good response and 2.0 ± 0.2 for the 5 patients with poor response) in lung cancer could be detected on early Tc-MIBI chest SPECT. The difference was significant (p < 0.05) by an independent Student t test. However, no significant differences were found for other prognostic factors (age, sex, body weight loss, performance status, tumor cell type, and tumor stage) between the good and poor response groups. Conclusion: Early Tc-MIBI chest SPECT can be used to understand the Pgp expression in NSCLC and to quickly predict chemotherapy response to paclitaxel.