This study evaluated the clinical role of Tc-99m-methoxyisobtylisonitrile (Tc-MIBI) single photon emission computed tomography (SPECT) of the chest in the detection of mediastinal lymph node (MLN) metastasis in patients with non-small cell lung cancer (NSCLC). Twenty-five patients with proven NSCLC were enrolled in this study. Each of the patients received computed tomography (CT) of the chest and Tc-MIBI SPECT of the chest for presurgical staging. A postsurgical pathologic diagnosis was made and these patients were evaluated for the study of mediastinal lymph nde (MLN) involvement. Meanwhile, 10 volunteers also accepted Tc-MIBI SPECT of the chest for comparison. The results showed that the diagnostic sensitivity, specificity and accuracy of Tc-MIBI chest SPECT were 81.8%, 85.7% and 84% and for chest CT they are 36.3%, 85.7% and 64%, respectively. Our results indicated that Tc-MIBI chest SPECT was more sensitive and accurate than chest CT in the evaluation and detection of MLN involvement in the NSCLC patients.