We investigated whether the combined use of computed tomography, thallium-201 single photon emission computed tomography and serum carcinoembryonic antigen level improves preoperative non-invasive mediastinal. 128 consecutive non-small cell lung cancer patients (85 adenocarcinomas, 31 squamous cell carcinomas and 12 others) who underwent a surgical resection were enrolled in this study. The results of the combined procedures were compared with the pathologic findings. Our results showed that the combined evaluation of mediastinal nodal involvement with the three procedures might increase underestimation, but decrease overestimation as compared to computed tomography alone. Thallium-201 single photon emission computed tomography for patients with enlarged nodes at computed tomography showed 81.3% and 100% of positive predictive value in overall and squamous cell carcinoma patients, respectively. The negative predictive value of thallium-201 single photon emission computed tomography for patients without enlarged nodes at computed tomography was highly accurate in adenocarcinoma (93.9%) as well as squamous cell carcinoma (94.4%). Combining computed tomography findings and serum carcinoembryonic antigen level had a poor predictive value. However, in patients with adenocarcinoma, a negative examination was highly accurate (95.2%). In conclusion, our results show a trend that combined use of the three procedures might improve non-invasive mediastinal staging in non-small cell lung cancer.