Confocal laser endomicroscopy is a novel endoscopic technique that may allow imaging of living cells in lung tissue in vivo. Here, we assessed the potential of this technique for detection of histology during screening bronchoscopy for lung cancer.Thirty-two patients with suspected malignancies underwent bronchoscopy with endomicroscopy using acriflavine hydrochloride. Standardized areas and localized lesions were analysed by in vivo confocal imaging during bronchoscopy and biopsies were taken. Confocal images were graded and correlated prospectively with conventional histology from biopsies.Acriflavine hydrochloride yielded high-quality confocal images and strongly labelled airway epithelial cells. No side effects were noted. Prospectively, 75,522 confocal images from 56 different locations were compared prospectively with histological data from biopsy specimens. Endomicroscopy allowed subsurface imaging with detailed analysis of cellular and subcellular structures. Neoplastic changes could be predicted with high accuracy (sensitivity, 96.0%; specificity, 87.1%; accuracy, 91.0%).Confocal laser endomicroscopy with acriflavine is a novel diagnostic tool for the analysis of living cells during bronchoscopy and permits virtual histology of neoplastic changes in the airways with high accuracy. This technique may enable the rapid diagnosis of neoplasia during ongoing endoscopy in patients with suspected lung cancer.