We developed a bronchoscope insertion guidance system that produces virtual images by extracting the bronchi by automatic threshold adjustment, and searching for the bronchial route to the determined target. We used this system in combination with a thin bronchoscope and endobronchial ultrasonography with a guide sheath (EBUS-GS), and evaluated its practicability, usefulness and safety. The subjects were 31 patients with 32 peripheral pulmonary lesions. Computed tomography (CT) data were transferred into this system, and virtual bronchial images were automatically produced by setting the lesion as the target. While virtual images with the target were displayed for comparison with real images by the system, a thin bronchoscope was advanced to the target bronchus. Transbronchial biopsy (TBB) was then performed by EBUS-GS. The system automatically produced virtual images to a median of fifth- (third- to seventh-) order bronchi. In all patients, the thin bronchoscope could be guided along the planned route, and observation to a median of fifth- (third- to seventh-) order bronchi was possible. Thirty lesions (93.8%) were successfully visualized by EBUS, and 27 (84.4%) could be pathologically diagnosed. In lesions < or =30mm in size, the EBUS visualization yield was 91.7% (22/24), and the diagnostic yield was 79.2% (19/24). The median total examination time was 22.3 (9.8-41.5) min. In summary, using the bronchoscope insertion guidance system, virtual images can be readily produced, and the bronchoscope can be successfully guided to the target. This method is promising as a routine examination method in the biopsy of peripheral pulmonary lesions.